Death domain containing receptor 4 antibodies and methods

ABSTRACT

The present invention relates to novel Death Domain Containing Receptor-4 (DR4) proteins which are members of the tumor necrosis factor (TNF) receptor family. In particular, isolated nucleic acid molecules are provided encoding the human DR4 proteins. DR4 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of DR4 activity and methods for using DR4 polynucleotides and polypeptides. The invention also relates to the treatment of diseases associated with reduced or increased levels of apoptosis using antibodies specific for DR4, which may be agonists and/or antagonists of DR4 activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)of provisional Application Nos. 60/990,687 and 60/885,971, filed Nov.28, 2007 and Jan. 22, 2007, respectively. This application is also aContinuation-in-Part and claims benefit of priority under 35 U.S.C. §120of non-provisional application Ser. No. 11/076,187, filed Mar. 10, 2005(now U.S. Pat. No. 7,476,384), which claims the benefit of priorityunder 35 U.S.C. §119(e) of provisional Application Nos. 60/551,768 and60/608,469, filed Mar. 11, 2004 and Sep. 10, 2004, respectively. Thisapplication is also a Continuation-in-Part and claims benefit ofpriority under 35 U.S.C. §120 of non-provisional application Ser. No.10/648,786, filed Aug. 27, 2003 (now U.S. Pat. No. 7,452,538), whichclaims the benefit of priority under 35 U.S.C. §119(e) of provisionalApplication Nos. 60/413,861 and 60/406,922, filed Sep. 27, 2002 and Aug.30, 2002 respectively. Application Ser. No. 10/648,786 is in turn aContinuation-In-Part and claims benefit of priority under 35 U.S.C. §120of non-provisional application Ser. No. 09/565,918, filed on May 5, 2000(now U.S. Pat. No. 6,433,147), which in turn claims the benefit ofpriority under 35 U.S.C. §119(e) of provisional Application No.60/132,922, filed May 6, 1999, and is a Continuation-In-Part claimingbenefit of priority under 35 U.S.C. §120 of non-provisional applicationSer. No. 09/013,895, filed on Jan. 27, 1998 (now U.S. Pat. No.6,342,363), which in turn claims the benefit of priority under 35 U.S.C.§119(e) of provisional Application Nos. 60/037,829 and 60/035,722, filedFeb. 5, 1997 and Jan. 28, 1997 respectively. Each of the above-citedapplications is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel member of the tumor necrosisfactor family of receptors. More specifically, isolated nucleic acidmolecules are provided encoding human Death Domain Containing Receptor4, sometimes herein “DR4”. DR4 polypeptides are also provided, as arevectors, host cells and recombinant methods for producing the same. Theinvention relates to the treatment of diseases associated with reducedor increased levels of apoptosis using antibodies specific for DR4,which may be agonists and/or antagonists of DR4 activity. The inventionfurther relates to screening methods for identifying agonists andantagonists of DR4 activity and methods for using DR4 polynucleotidesand polypeptides.

RELATED ART

Many biological actions, for instance, response to certain stimuli andnatural biological processes, are controlled by factors, such ascytokines. Many cytokines act through receptors by engaging the receptorand producing an intra-cellular response.

For example, tumor necrosis factors (TNF) alpha and beta are cytokineswhich act through TNF receptors to regulate numerous biologicalprocesses, including protection against infection and induction of shockand inflammatory disease. The TNF molecules belong to the “TNF-ligand”superfamily, and act together with their receptors or counter-ligands,the “TNF-receptor” superfamily. So far, nine members of the TNF ligandsuperfamily have been identified and ten members of the TNF-receptorsuperfamily have been characterized.

Among the ligands there are included TNF-α, lymphotoxin-α (LT-α, alsoknown as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL,CD40L, CD27L, CD30L, 4-IBBL, OX40L and nerve growth factor (NGF). Thesuperfamily of TNF receptors includes the p55TNF receptor, p75TNFreceptor, TNF receptor-related protein, FAS antigen or APO-1, CD40,CD27, CD30, 4-IBB, OX40, low affinity p75 and NGF-receptor (Meager, A.,Biologicals, 22:291-295 (1994)).

Many members of the TNF-ligand superfamily are expressed by activatedT-cells, implying that they are necessary for T-cell interactions withother cell types which underlie cell ontogeny and functions. (Meager,A., supra).

Considerable insight into the essential functions of several members ofthe TNF receptor family has been gained from the identification andcreation of mutants that abolish the expression of these proteins. Forexample, naturally occurring mutations in the FAS antigen and its ligandcause lymphoproliferative disease (Watanabe-Fukunaga, R., et al., Nature356:314 (1992)), perhaps reflecting a failure of programmed cell death.Mutations of the CD40 ligand cause an X-linked immunodeficiency statecharacterized by high levels of immunoglobulin M and low levels ofimmunoglobulin G in plasma, indicating faulty T-cell-dependent B-cellactivation (Allen, R. C. et al., Science 259:990 (1993)). Targetedmutations of the low affinity nerve growth factor receptor cause adisorder characterized by faulty sensory innovation of peripheralstructures (Lee, K. F. et al., Cell 69:737 (1992)).

TNF and LT-α are capable of binding to two TNF receptors (the 55- and75-kd TNF receptors). A large number of biological effects elicited byTNF and LT-α, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT-α are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B.and Von Huffel, C., Science 264:667-668 (1994)). Mutations in the p55Receptor cause increased susceptibility to microbial infection.

Moreover, an about 80 amino acid domain near the C-terminus of TNFR1(p55) and Fas was reported as the “death domain,” which is responsiblefor transducing signals for programmed cell death (Tartaglia et al.,Cell 74:845 (1993)).

Apoptosis, or programmed cell death, is a physiologic process essentialto the normal development and homeostasis of multicellular organisms (H.Steller, Science 267, 1445-1449 (1995)). Derangements of apoptosiscontribute to the pathogenesis of several human diseases includingcancer, neurodegenerative disorders, and acquired immune deficiencysyndrome (C. B. Thompson, Science 267, 1456-1462 (1995)). Recently, muchattention has focused on the signal transduction and biological functionof two cell surface death receptors, Fas/APO-1 and TNFR-1 (J. L.Cleveland, et al., Cell 81, 479-482 (1995); A. Fraser, et al., Cell 85,781-784 (1996); S, Nagata, et al., Science 267, 1449-56 (1995)). Bothare members of the TNF receptor family which also include TNFR-2, lowaffinity NGFR, CD40, and CD30, among others (C. A. Smith, et al.,Science 248, 1019-23 (1990); M. Tewari, et al., in Modular Texts inMolecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman andHall, London, 1995). While family members are defined by the presence ofcysteine-rich repeats in their extracellular domains, Fas/APO-1 andTNFR-1 also share a region of intracellular homology, appropriatelydesignated the “death domain”, which is distantly related to theDrosophila suicide gene, reaper (P. Golstein, et al., Cell 81, 185-6(1995); K. White et al., Science 264, 677-83 (1994)). This shared deathdomain suggests that both receptors interact with a related set ofsignal transducing molecules that, until recently, remainedunidentified. Activation of Fas/APO-1 recruits the deathdomain-containing adapter molecule, FADD/MORT1 (A. M. Chinnaiyan, etal., Cell 81, 505-12 (1995); M. P. Boldin, et al., J. Biol Chem 270,7795-8 (1995); F. C. Kischkel, et al., EMBO 14, 5579-5588 (1995)), whichin turn binds and presumably activates FLICE/MACH1, a member of theICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al., Cell 85,817-827 (1996); M. P. Boldin, et al., Cell 85, 803-815 (1996)). Whilethe central role of Fas/APO-1 is to trigger cell death, TNFR-1 cansignal an array of diverse biological activities-many of which stem fromits ability to activate NF-kB (L. A. Tartaglia, et al., Immunol Today13, 151-3 (1992)). Accordingly, TNFR-1 recruits the multivalent adaptermolecule TRADD, which like FADD, also contains a death domain (H. Hsu,et al., Cell 81, 495-504 (1995); H. Hsu, et al., Cell 84, 299-308(1996)). Through its associations with a number of signaling moleculesincluding FADD, TRAF2, and RIP, TRADD can signal both apoptosis andNF-kB activation (H. Hsu, et al., Cell 84, 299-308 (1996); H. Hsu, etal., Immunity 4, 387-396 (1996)).

Recently a new apoptosis inducing ligand was discovered. Wiley, S. R. etal., refer to the new molecule as TNF-related apoptosis-inducing ligandor (“TRAIL”) (Immunity 3:673-682 (1995)). Pitti, R. M. et al., refer tothe new molecule as Apo-2 ligand or (“Apo-2L”). For convenience, it willbe referred to herein as TRAIL.

Unlike FAS ligand whose transcripts appear to be largely restricted tostimulated T-cells, significant levels of TRAIL are seen in manytissues, and it is constitutively transcribed by some cell lines. It hasbeen shown that TRAIL acts independently from FAS ligand (Wiley, S. R.,et al. (1995)), supra). Studies by Marsters, S. A. et al., haveindicated that TRAIL activates apoptosis rapidly, within a time framethat is similar to death signaling by FAS/Apo-1L but much faster thanTNF-induced apoptosis (Current Biology, 6:750-752 (1996)). All work todate suggest that the receptor for TRAIL is not one of the many knownTNF-receptors.

The effects of TNF family ligands and TNF family receptors are variedand influence numerous functions, both normal and abnormal, in thebiological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of such receptors andligands that influence biological activity, both normally and in diseasestates. In particular, there is a need to isolate and characterize thereceptor for the newly discovered TRAIL ligand.

SUMMARY OF THE INVENTION

The present invention provides for isolated nucleic acid moleculescomprising, or alternatively consisting of, nucleic acid sequencesencoding the amino acid sequence shown in SEQ ID NO:2 or the amino acidsequence encoding the cDNA clone deposited as ATCC Deposit No. 97853 onJan. 21, 1997.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as to methods of making such vectors and host cells and forusing them for production of DR4 polypeptides or peptides by recombinanttechniques.

The invention further provides an isolated DR4 polypeptide having anamino acid sequence encoded by a polynucleotide described herein.

The present invention also provides diagnostic assays such asquantitative and diagnostic assays for detecting levels of DR4 protein.Thus, for instance, a diagnostic assay in accordance with the inventionfor detecting over-expression of DR4, or soluble form thereof, comparedto normal control tissue samples may be used to detect the presence oftumors. See, for example, the assays described in Example 29.

Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes.Cellular response to TNF-family ligands include not only normalphysiological responses, but also diseases associated with increasedapoptosis or the inhibition of apoptosis. Apoptosis-programmed celldeath-is a physiological mechanism involved in the deletion ofperipheral T lymphocytes of the immune system, and its dysregulation canlead to a number of different pathogenic processes. Diseases associatedwith increased cell survival, or the inhibition of apoptosis, includecancers, autoimmune disorders, viral infections, inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection.Diseases associated with increased apoptosis include AIDS,neurodegenerative disorders, myelodysplastic syndromes, ischemic injury,toxin-induced liver disease, septic shock, cachexia and anorexia.

Thus, the invention further provides a method for enhancing apoptosisinduced by a TNF-family ligand, which involves administering to a cellwhich expresses the DR4 polypeptide an effective amount of an agonistcapable of increasing DR4 mediated signaling. Preferably, DR4 mediatedsignaling is increased to treat and/or prevent a disease whereindecreased apoptosis is exhibited.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the DR4 polypeptide an effectiveamount of an antagonist capable of decreasing DR4 mediated signaling.Preferably, DR4 mediated signaling is decreased to treat and/or preventa disease wherein increased apoptosis is exhibited.

The present invention relates to the detection, diagnosis, prognosisand/or treatment of diseases and disorders of cell death, including butnot limited to cancers, using compositions comprising polynucleotidesencoding DR4, the polypeptides encoded by these polynucleotides andantibodies that immunospecifically bind these polypeptides. Theinvention further relates to diagnostic and therapeutic methods usefulfor diagnosing, treating, preventing and/or prognosing disorders of celldeath, and therapeutic methods for treating such disorders. Theinvention further relates to screening methods for identifying agonistsand antagonists of polynucleotides and polypeptides of the invention.The invention further relates to methods and/or compositions forinhibiting or promoting the production and/or function of thepolypeptides of the invention. The invention is based in part on theability of DR4 to stimulate apoptosis and thus prevent tumorprogression, as demonstrated in Examples 5 and 6, below.

In accordance with one embodiment of the present invention, there isprovided an isolated antibody that binds specifically to a DR4polypeptide, as well as biologically active fragments, analogs andderivatives thereof, together with fragments, analogs and derivativesthereof which may be useful in the diagnosis or treatment of diseases ordisorders associated with decreased levels of cell death.

In one preferred embodiment of the present invention is presented anisolated antibody which is an agonist of DR4 activity and therefore maybe useful in the treatment of diseases or disorders associated withdecreased levels of cell death including, for example, prostate,pancreatic, hepatic, lung, breast, ovarian, colorectal and hematologicalcancers.

In accordance with another embodiment of the present invention, there isprovided an isolated antibody that binds specifically to a DR4polypeptide, as well as biologically active fragments, analogs andderivatives thereof, together with fragments, analogs and derivativesthereof which may be useful in the diagnosis or treatment of diseases ordisorders associated with increased levels of cell death.

In another preferred embodiment of the present invention is presented anisolated antibody which is an antagonist of DR4 activity and thereforemay be useful in the treatment of diseases or disorders associated withincreased levels of cell death including, for example, myelodysplasticsyndrome.

The present invention also provides pharmaceutical compositionscomprising DR4 antibodies, as described above, which may be used forinstance, to treat, prevent, prognose and/or diagnose diseases ordisorders associated with abnormal levels of cell death and/orconditions associated with such diseases or disorders.

In preferred embodiments the present invention provides pharmaceuticalcompositions comprising DR4 agonistic antibodies, which may be used forinstance to treat, prevent, prognose and/or diagnose diseases ordisorders associated with increased or decreased levels of cell death aswell as conditions associated with such diseases or disorders.

Whether any candidate “agonist” or “antagonist” of the present inventioncan enhance or inhibit apoptosis can be determined using art-knownTNF-family ligand/receptor cellular response assays, including thosedescribed in more detail below. Thus, in a further aspect, a screeningmethod is provided for determining whether a candidate agonist orantagonist is capable of enhancing or inhibiting a cellular response toa TNF-family ligand. The method involves contacting cells which expressthe DR4 polypeptide with a candidate compound and a TNF-family ligand,assaying a cellular response, and comparing the cellular response to astandard cellular response, the standard being assayed when contact ismade with the ligand in absence of the candidate compound, whereby anincreased cellular response over the standard indicates that thecandidate compound is an agonist of the ligand/receptor signalingpathway and a decreased cellular response compared to the standardindicates that the candidate compound is an antagonist of theligand/receptor signaling pathway. By the invention, a cell expressingthe DR4 polypeptide can be contacted with either an endogenous orexogenously administered TNF-family ligand.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide and deduced amino acid sequence of DR4 (SEQID NOS: 1 and 2, respectively). It is predicted that amino acids fromabout 1 to about 23 constitute the signal peptide, amino acids fromabout 24 to about 238 constitute the extracellular domain, amino acidsfrom about 131 to about 229 constitute the cysteine rich domain, aminoacids from about 239 to about 264 constitute the transmembrane domain,and amino acids from about 265 to about 468 constitute the intracellulardomain of which amino acids from about 379 to about 422 constitute thedeath domain.

FIG. 2 shows the regions of similarity between the amino acid sequencesof DR4 (SEQ ID NO:2), human tumor necrosis factor receptor 1 (SEQ IDNO:3), human Fas protein (SEQ ID NO:4), and the death domain containingreceptor 3 (DR3) (SEQ ID NO:5). Residues that match the consensus areshaded.

FIG. 3 shows an analysis of the DR4 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown, as predicted for the amino acid sequence depicted in FIG. 1 (SEQID NO:2) using the default parameters of the recited computer programs.In the “Antigenic Index—Jameson-Wolf” graph, amino acid residues 35-92,114-160, 169-240, 267-298, 330-364, 391-404, and 418-465 in FIG. 1 (SEQID NO:2) correspond to the shown highly antigenic regions of the DR4protein.

FIG. 4 shows the nucleotide sequences of related nucleic acid fragmentsHTOIY07R (SEQ ID NO:6) and HTXEY80R (SEQ ID NO:7).

FIGS. 5A and 5B show the ability of DR4 to induce apoptosis in the celllines MCF7 and 293. FIG. 5C shows the ability of death proteaseinhibitors z-VAD-fmk and CrmA to inhibit the apoptotic action of DR4.

FIG. 6A shows the ability of a soluble extracellular DR4-Fc fusion toblock the apoptotic inducing ability of TRAIL.

FIG. 6B shows the inability of soluble extracellular DR4-Fc fusion toblock the apoptotic inducing ability of TNF-alpha.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated nucleic acid moleculescomprising/, or alternatively consisting of, a nucleic acid sequenceencoding the DR4 polypeptide whose amino acid sequence is shown in SEQID NO:2, or a fragment of the polypeptide. The DR4 polypeptide of thepresent invention shares sequence homology with human TNFR-I, DR3 andFas ligand (FIG. 2). The nucleotide sequence shown in SEQ ID NO:1 wasobtained by sequencing cDNA clones such as HCUDS60, which was depositedon Jan. 21, 1997 at the American Type Culture Collection, 10801University Boulevard, Manassas, Va., 20110-2209, and given AccessionNumber 97853. The deposited clone is contained in the pBK plasmid(Stratagene, La Jolla, Calif.).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been substantially purified by anysuitable technique such as, for example, the single-step purificationmethod disclosed in Smith and Johnson, Gene 67:31-40 (1988).

Using the information provided herein, such as the nucleic acid sequenceset out in SEQ ID NO: 1, a nucleic acid molecule of the presentinvention encoding a DR4 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the gene ofthe present invention has also been identified in cDNA libraries of thefollowing tissues: amniotic cells, heart, liver cancer, kidney,leukocyte, activated T-cell, K562 plus PMA, W138 cells, Th2 cells, humantonsils, and CD34 depleted buffy coat (cord blood).

The DR4 gene contains an open reading frame encoding a mature protein ofabout 445 amino acid residues whose initiation codon is at position19-21 of the nucleotide sequence shown in SEQ ID NO.1, with a leadersequence of about 23 amino acid residues (i.e., a total protein lengthof 468 amino acids), and a deduced molecular weight of about 50 kDa. Inthis context “about” includes the particularly recited size, larger orsmaller by several (5, 4, 3, 2, or 1) amino acid residues, at eitherterminus or at both termini.

Of known members of the TNF receptor family, the DR4 polypeptide of theinvention shares the greatest degree of homology with human TNFR1 andDR3 polypeptides shown in FIG. 2, including significant sequencehomology over the multiple Cysteine Rich domains.

In addition to the sequence homology exhibited between DR4 and otherdeath domain containing receptors, DR4 has been shown to bind to TRAILand to induce apoptosis when transiently expressed. MCF7 human breastcarcinoma cells and 293 cells were transiently transfected with a DR4expressing construct, as described in Example 5. As shown in FIGS. 5Aand 5B a substantial proportion of transfected cells underwent themorphological changes characteristic of apoptosis. As anticipated,deletion of the death domain abolished the ability of DR4 to engage thedeath pathway. As can be seen in FIG. 5C, DR4-induced apoptosis wasefficiently blocked by inhibitors of death proteases includingz-VAD-fmk, an irreversible broad spectrum caspase inhibitor and CrmA, acowpox virus encoded serpin that preferentially inhibits apical caspasessuch as FLICE/MACH-1 (caspase-8). Since TNFR-1, CD-95 and DR3-inducedapoptosis is also attenuated by these same inhibitors, it is likely thatthe downstream death effector molecules are similar in nature.

To determine if DR4 was capable of binding TRAIL, the extracellularligand binding domain of DR4 was expressed as a fusion to the Fc regionof human IgG (DR4-Fc). TRAIL selectively bound to DR4-Fc but not tocorresponding extracellular domains of TNFR-1 or CD-95, also expressedas Fc fusions, data not shown. Additionally, DR4-Fc did not bind eitherTNF alpha or Fas ligand under conditions where both of these ligandsbound their cognate receptors.

The ability of TRAIL to induce apoptosis in MCF7 cells was specificallyblocked by DR4-Fc but not influenced by TNFR1-Fc, CD95-Fc or Fc alone(FIG. 6A). Further, as expected, TNF alpha-induced apoptosis wasinhibited by TNFR-1-Fc but not by DR4-Fc, CD95-Fc or Fc alone (FIG. 6B).

Taken together, the data described above indicate that DR4 is a deathdomain containing receptor with the ability to induce apoptosis and is areceptor for TRAIL a known apoptosis inducing ligand.

As indicated, the present invention also provides the mature form(s) ofthe DR4 protein of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides a nucleotide sequence encodingthe mature DR4 polypeptide having the amino acid sequence encoded by thecDNA contained in the host identified as ATCC Deposit No. 97853, and asshown in SEQ ID NO:2. By the mature DR4 protein having the amino acidsequence encoded by the cDNA contained in the host identified as ATCCDeposit No. 97853, is meant the mature form(s) of the DR4 proteinproduced by expression in a mammalian cell (e.g., COS cells, asdescribed below) of the complete open reading frame encoded by the humancDNA contained in the vector in the deposited host. As indicated below,the mature DR4 having the amino acid sequence encoded by the cDNAcontained in ATCC Deposit No. 97853, may or may not differ from thepredicted “mature” DR4 protein shown in SEQ ID NO:2 (amino acids fromabout 24 to about 468 in SEQ ID NO:2) depending on the accuracy of thepredicted cleavage site based on computer analysis. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) amino acid residues, at either terminus or atboth termini.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the method of McGeoch (Virus Res. 3:271-286 (1985)) and vonHeinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequence of the completeDR4 polypeptide of the present invention was analyzed by a computerprogram (“PSORT”). (See K. Nakai and M. Kanehisa, Genomics 14:897-911(1992)), which is an expert system for predicting the cellular locationof a protein based on the amino acid sequence. As part of thiscomputational prediction of localization, the methods of McGeoch and vonHeinje are incorporated. The analysis by the PSORT program predicted thecleavage sites between amino acids 23 and 24 in SEQ ID NO:2. Thereafter,the complete amino acid sequences were further analyzed by visualinspection, applying a simple form of the (−1, −3) rule of von Heine.von Heinje, supra. Thus, the leader sequence for the DR4 protein ispredicted to consist of amino acid residues 1-23, underlined in SEQ IDNO:2, while the predicted mature DR4 protein consists of about residues24-468.

As one of ordinary skill would appreciate, due to the possibility ofsequencing errors, as well as the variability of cleavage sites forleaders in different known proteins, the predicted DR4 receptorpolypeptide encoded by the deposited cDNA comprises about 468 aminoacids, but may be anywhere in the range of 458478 amino acids; and thepredicted leader sequence of this protein is about 40 amino acids, butmay be anywhere in the range of about 30 to about 50 amino acids. Itwill further be appreciated that, the domains described herein have beenpredicted by computer analysis, and accordingly, that depending on theanalytical criteria used for identifying various functional domains, theexact “address” of, for example, the extracelluar domain, intracellulardomain, death domain, cysteine-rich motifs, and transmembrane domain ofDR4 may differ slightly. For example, the exact location of the DR4extracellular domain in SEQ ID NO:2 may vary slightly (e.g., the addressmay “shift” by about 1 to about 20 residues, more likely about 1 toabout 5 residues) depending on the criteria used to define the domain.In this context “about” includes the particularly recited size, largeror smaller by several (5, 4, 3, 2, or 1) amino acid residues, at eitherterminus or at both termini. In any event, as discussed further below,the invention further provides polypeptides having various residuesdeleted from the N-terminus and/or C-terminus of the complete DR4,including polypeptides lacking one or more amino acids from theN-termini of the extracellular domain described herein, which constitutesoluble forms of the extracellular domain of the DR4 polypeptides.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution.

However, a nucleic acid molecule contained in a clone that is a memberof a mixed clone library (e.g., a genomic or cDNA library) and that hasnot been isolated from other clones of the library (e.g., in the form ofa homogeneous solution containing the clone without other members of thelibrary) or a chromosome isolated or removed from a cell or a celllysate (e.g., a “chromosome spread”, as in a karyotype), is not“isolated” for the purposes of this invention. Isolated RNA moleculesinclude in vivo or in vitro RNA transcripts of the DNA molecules of thepresent invention. Isolated nucleic acid molecules according to thepresent invention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DR4DNAmolecules comprising, or alternatively consisting of, an open readingframe (ORF) shown in SEQ ID NO:1 and further include DNA molecules whichcomprise, or alternatively consist of, a sequence substantiallydifferent than all or part of the ORF whose initiation codon is atposition 19-21 of the nucleotide sequence shown in SEQ ID NO:1 butwhich, due to the degeneracy of the genetic code, still encode the DR4polypeptide or a fragment thereof. Of course, the genetic code is wellknown in the art. Thus, it would be routine for one skilled in the artto generate such degenerate variants.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the DR4 polypeptide having an amino acid sequenceencoded by the cDNA contained in the plasmid deposited as ATCC DepositNo. 97853 on Jan. 21, 1997. Preferably, these nucleic acid moleculeswill encode the mature polypeptide encoded by the above-describeddeposited cDNA. The invention further provides an isolated nucleic acidmolecule having the nucleotide sequence shown in SEQ ID NO:1 or thenucleotide sequence of the DR4 cDNA contained in the above-describeddeposited plasmid, or a nucleic acid molecule having a sequencecomplementary to one of the above sequences. Such isolated DNA moleculesand fragments thereof, have uses which include, but are not limited to,as DNA probes for gene mapping by in situ hybridization of the DR4 genein human tissue by Northern blot analysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By fragments of an isolated DNAmolecule having the nucleotide sequence shown in SEQ ID NO:1 or havingthe nucleotide sequence of the deposited cDNA (the cDNA contained in theplasmid deposited as ATCC Deposit No. 97853) are intended DNA fragmentsat least 20 nt, and more preferably at least 30 nt in length, and evenmore preferably, at least about 40, 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 nt in length,which are useful as DNA probes as discussed above. Of course, DNAfragments corresponding to most, if not all, of the nucleotide sequenceshown in SEQ ID NO:1 are also useful as DNA probes. By a fragment about20 nt in length, for example, is intended fragments which include 20 ormore bases from the nucleotide sequence in SEQ ID NO:1. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini.

Representative examples of DR4 polynucleotide fragments of the inventioninclude, for example, fragments that comprise, or alternatively consistof, a sequence from about nucleotide 19 to 87, 88 to 732, 88 to 138, 139to 189, 190 to 240, 241 to 291, 292 to 342, 343 to 705, 343 to 393, 394to 444, 445 to 495, 496 to 546, 547 to 597, 598 to 648, 649 to 699, 700to 732, 733 to 810, 733 to 771, 772 to 810, 811 to 1422, 811 to 861, 862to 912, 913 to 963, 964 to 1014, 1015 to 1065, 1066 to 1116, 1117 to1167, 1153 to 1284, 1153 to 1203, 1204 to 1254, 1255 to 1284, 1168 to1218, 1219 to 1269, 1270 to 1320, 1321 to 1371, and 1372 to 1422 of SEQID NO:1, or the complementary strand thereto, or the cDNA contained inthe deposited plasmid. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini.

The present invention is further directed to polynucleotides comprising,or alternatively consisting of, isolated nucleic acid molecules whichencode domains of DR4. In one aspect, the invention providespolynucleotides comprising, or alternatively consisting of, nucleic acidmolecules which encode beta-sheet regions of DR4 protein set out inTable 1. Representative examples of such polynucleotides include nucleicacid molecules which encode a polypeptide comprising, or alternativelyconsisting of, one, two, three, four, five or more amino acid sequencesselected from the group consisting of amino acid residues from about 8to about 17, amino acid residues from about 53 to about 60, amino acidresidues from about 87 to about 103, amino acid residues from about 146to about 155, amino acid residues from about 161 to about 166, aminoacid residues from about 214 to about 221, amino acid residues fromabout 240 to about 252, amino acid residues from about 257 to about 264,amino acid residues from about 274 to about 283, amino acid residuesfrom about 324 to about 329, amino acid residues from about 349 to about354, amino acid residues from about 363 to about 369, amino acidresidues from about 371 to about 376, amino acid residues from about 394to about 399, and amino acid residues from about 453 to about 458 in SEQID NO:2. In this context “about” includes the particularly recited valueand values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.Polypeptides encoded by these polynucleotides are also encompassed bythe invention.

In specific embodiments, the polynucleotide fragments of the inventionencode a polypeptide which demonstrates a DR4 functional activity. By apolypeptide demonstrating a DR4 “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a complete (full-length) or mature DR4polypeptide. Such functional activities include, but are not limited to,biological activity (e.g., ability to induce apoptosis in cellsexpressing the polypeptide (see, e.g., Example 5), antigenicity (abilityto bind (or compete with a DR4 polypeptide for binding) to an anti-DR4antibody), immunogenicity (ability to generate antibody which binds to aDR4 polypeptide), ability to form multimers, and ability to bind to areceptor or ligand for a DR4 polypeptide (e.g., TRAIL; Wiley et al.,Immunity 3, 673-682 (1995)).

The functional activity of DR4 polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length (complete) DR4 polypeptide for bindingto anti-DR4 antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a DR4 ligand is identified (e.g., TRAIL),or the ability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E., et al., Microbiol. Rev.59:94-123 (1995). In another embodiment, physiological correlates of DR4binding to its substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples 5 and 6), and thoseotherwise known in the art may routinely be applied to measure theability of DR4 polypeptides and fragments, variants derivatives, andanalogs thereof to elicit DR4 related biological activity (e.g., abilityto bind TRAIL (see e.g., Example 6), ability to induce apoptosis incells expressing the polypeptide (see e.g., Example 5) in vitro or invivo). For example, biological activity can routinely be measured usingthe cell death assays performed essentially as previously described(Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol.Chem. 270:7795-8 (1995); Kischkel et al., EMBO 14:5579-5588 (1995);Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996)) and as set forthin Example 5 below. In one embodiment involving MCF7 cells, plasmidsencoding full-length DR4 or a candidate death domain containing receptorare co-transfected with the pLantern reporter construct encoding greenfluorescent protein. Nuclei of cells transfected with DR4 will exhibitapoptotic morphology as assessed by DAPI staining.

Other methods will be known to the skilled artisan and are within thescope of the invention.

Preferred nucleic acid fragments of the present invention include anucleic acid molecule encoding a member selected from the group: apolypeptide comprising, or alternatively consisting of, the DR4extracellular domain (amino acid residues from about 24 to about 238 inSEQ ID NO:2); a polypeptide comprising, or alternatively consisting of,the DR4 cysteine rich domain (amino acid residues from about 131 toabout 229 in SEQ ID NO:2); a polypeptide comprising, or alternativelyconsisting of, the DR4 transmembrane domain (amino acid residues fromabout 239 to about 264 in SEQ ID NO:2); a fragment of the predictedmature DR4 polypeptide, wherein the fragment has a DR4 functionalactivity (e.g., antigenic activity or biological activity); apolypeptide comprising, or alternatively consisting of, the DR4intracellular domain (amino acid residues from about 265 to about 468 inSEQ ID NO:2); a polypeptide comprising, or alternatively consisting of,the DR4 receptor extracellular and intracellular domains with all orpart of the transmembrane domain deleted; a polypeptide comprising, oralternatively consisting of, DR4 receptor death domain (predicted toconstitute amino acid residues from about 379 to about 422 in SEQ IDNO:2); a polypeptide comprising, or alternatively consisting of, one,two, three, four or more, epitope bearing portions of the DR4 receptorprotein. In additional embodiments, the polynucleotide fragments of theinvention encode a polypeptide comprising, or alternatively consistingof, any combination of 1, 2, 3, 4, 5, 6, 7, or all 8 of theabove-encoded polypeptide embodiments. As above, with the leadersequence, the amino acid residues constituting the DR4 receptorextracellular, transmembrane and intracellular domains have beenpredicted by computer analysis. Thus, one of ordinary skill wouldappreciate that the amino acid residues constituting these domains mayvary slightly (e.g., by about 1 to 15 residues) depending on thecriteria used to define the domain. Polypeptides encoded by thesenucleic acid molecules are also encompassed by the invention.

It is believed one or both of the extracellular cysteine rich motifs ofthe DR4 polypeptide disclosed in SEQ ID NO:2 is important forinteractions between DR4 and its ligands (e.g., TRAIL). Accordingly,specific embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, theamino acid sequence of one or both of amino acid residues 131 to 183,and/or 184 to 229 of SEQ ID NO:2. In a specific embodiment thepolynucleotides encoding DR4 polypeptides of the invention comprise, oralternatively consist of both of the extracellular cysteine rich motifsdisclosed in SEQ ID NO:2. Polypeptides encoded by these polynucleotidesare also encompassed by the invention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of DR4. Preferred embodiments of the invention inthis regard include fragments that comprise, or alternatively consistof, one, two, three, four, or more of the following functional domains:alpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of DR4.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table I). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table Iinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in SEQ IDNO:2. As set out in FIG. 3 and in Table I, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions (columns I, III, V, and VII in Table I), Chou-Fasmanalpha-regions, beta-regions, and turn-regions (columns II, IV, and VI inTable I), Kyte-Doolittle hydrophilic regions (column VIII in Table I),Hopp-Woods hydrophobic regions (column IX in Table I), Eisenberg alpha-and beta-amphipathic regions (columns X and XI in Table I),Karplus-Schulz flexible regions (column XII in Table I), Jameson-Wolfregions of high antigenic index (column XIII in Table I), and Eminisurface-forming regions (column XIV in Table I). Among highly preferredpolynucleotides in this regard are those that encode polypeptidescomprising, or alternatively consisting of, regions of DR4 that combineseveral structural features, such as several (e.g., 1, 2, 3, or 4) ofthe same or different region features set out above.

The data representing the structural or functional attributes of DR4 setforth in FIG. 3 and/or Table I, as described above, was generated usingthe various modules and algorithms of the DNA*STAR set on defaultparameters. In a preferred embodiment, the data presented in columnsVIII, XII, and XIII of Table I can be used to determine regions of DR4which exhibit a high degree of potential for antigenicity. Regions ofhigh antigenicity are determined from the data presented in columnsVIII, XII, and/or XIII by choosing values which represent regions of thepolypeptide which are likely to be exposed on the surface of thepolypeptide in an environment in which antigen recognition may occur inthe process of initiation of an immune response.

TABLE I Res Pos. I II III IV V VI VII VIII IX X XI XII XIII Met 1 . . B. . . . 0.12 . . . −0.10 0.90 Ala 2 . . . . . . C −0.08 * * . 0.25 1.08Pro 3 . . . . . . C 0.42 * * . 0.10 0.86 Pro 4 . . . . . T C −0.04 * * .1.05 1.69 Pro 5 A . . . . T . 0.31 . * F 1.00 1.24 Ala 6 A . . . . T .0.10 . * F 1.00 1.10 Arg 7 A . . . . T . 0.34 . * . 0.10 0.58 Val 8 . .B B . . . −0.03 . * . −0.30 0.37 His 9 . . B B . . . −0.52 . * . −0.300.37 Leu 10 . . B B . . . −1.12 . * . −0.60 0.17 Gly 11 . . B B . . .−1.12 . * . −0.60 0.18 Ala 12 . . B B . . . −2.09 . * . −0.60 0.14 Phe13 . . B B . . . −1.54 . * . −0.60 0.12 Leu 14 . . B B . . . −1.72 . . .−0.60 0.18 Ala 15 . . B B . . . −0.91 . . . −0.60 0.27 Val 16 . . B B .. . −0.78 . . . −0.60 0.51 Thr 17 . . B B . . . −0.53 . . F −0.45 0.95Pro 18 . . . B . . C −0.13 . . F 0.05 0.93 Asn 19 . . . . . T C 0.09 . .F 0.60 1.69 Pro 20 . . . . . T C 0.09 . . F 0.60 1.18 Gly 21 . . . . T T. 0.64 . . F 0.65 0.77 Ser 22 . . . . . T C 0.61 . . F 0.45 0.64 Ala 23. . . . . . C 0.51 . . F 0.25 0.41 Ala 24 . . . . . T C 0.51 . . F 0.450.60 Ser 25 . . B . . T . 0.13 . . F 0.85 0.78 Gly 26 A . . . . T .−0.11 . . F 0.85 0.78 Thr 27 A . . . . T . −0.40 . . F 0.85 0.78 Glu 28A A . . . . . −0.40 . . F 0.45 0.58 Ala 29 A A . . . . . −0.12 . . .0.30 0.60 Ala 30 A A . . . . . −0.03 . . . 0.30 0.60 Ala 31 A A . . . .. 0.01 . . . 0.30 0.53 Ala 32 A A . . . . . 0.37 . . . −0.30 0.71 Thr 33A . . . . T . −0.49 * . F 1.00 1.40 Pro 34 A . . . . T . −0.19 . . F1.00 1.03 Ser 35 . . B . . T . 0.06 . . F 0.40 1.07 Lys 36 . . B . . T .0.34 . . F 0.25 0.73 Val 37 . . B B . . . 0.63 . . F −0.15 0.64 Trp 38 .. B B . . . 0.36 . . F −0.15 0.64 Gly 39 . . B B . . . 0.22 * * F −0.150.32 Ser 40 . . . . . . C 0.63 * * F −0.05 0.43 Ser 41 . . . . . T C−0.30 * * F 0.45 0.80 Ala 42 . . . . . T C 0.56 * * F 1.05 0.57 Gly 43 .. . . . T C 0.63 * * F 1.35 0.73 Arg 44 . . B . . T . 1.09 * * F 1.490.84 Ile 45 . . B . . . . 1.04 * * F 1.78 1.63 Glu 46 . . B . . . .1.00 * * F 2.12 1.63 Pro 47 . . B . . T . 1.24 * * F 2.51 0.83 Arg 48 .. . . T T . 1.70 * * F 3.40 1.17 Gly 49 . . . . T T . 1.24 * * F 3.061.32 Gly 50 . . . . T T . 1.54 * * F 2.57 0.84 Gly 51 . . . . . T C0.73 * * F 2.03 0.44 Arg 52 . . . . . T C 0.73 * * F 1.39 0.36 Gly 53 .. B . . T . 0.31 * * F 0.85 0.57 Ala 54 . . B . . T . 0.36 . * F 0.850.83 Leu 55 . . B . . . . 0.10 . * F 0.65 0.57 Pro 56 . . B . . . . 0.10. * F −0.25 0.57 Thr 57 . . B . . . . −0.01 . * F −0.25 0.55 Ser 58 . .B . . T . 0.30 . . F 0.10 1.16 Met 59 . . B . . T . 0.54 . . F 0.40 1.02Gly 60 . . B . . T . 1.14 . . F 0.25 0.70 Gln 61 . . . . T T . 1.06 . .F 0.65 0.81 His 62 . . . . . . C 0.78 . * F 0.40 1.10 Gly 63 . . . . . TC 1.19 . * F 0.60 1.12 Pro 64 . . . . . T C 1.20 . * F 1.20 1.27 Ser 65. . . . . T C 1.66 . * F 1.05 0.94 Ala 66 . . B . . T . 1.07 . * F 1.301.86 Arg 67 . . B . . . . 0.76 * * . 1.29 1.22 Ala 68 . . B . . . .1.21 * * . 1.48 0.90 Arg 69 . . B . . T . 0.83 . * . 2.17 1.74 Ala 70 .. B . . T . 0.92 . * F 2.51 0.90 Gly 71 . . . . T T . 1.17 . * F 3.401.37 Arg 72 . . . . . T C 0.84 . * F 2.71 0.69 Ala 73 . . . . . T C1.54 * . F 2.48 1.06 Pro 74 . . . . . T C 1.22 * . F 2.70 2.10 Gly 75 .. . . . T C 1.22 * . F 2.62 1.66 Pro 76 . . . . . T C 1.68 * * F 2.241.66 Arg 77 . . . . . . C 1.57 * . F 2.60 2.10 Pro 78 . A B . . . .1.57 * . F 1.94 3.68 Ala 79 . A B . . . . 1.48 * . F 1.68 2.40 Arg 80 .A B . . . . 1.61 * * F 1.42 1.64 Glu 81 . A B . . . . 1.93 * * F 1.161.64 Ala 82 A A . . . . . 1.01 * * F 0.90 3.19 Ser 83 A . . . . T .1.33 * * F 1.30 1.34 Pro 84 A . . . . T . 1.07 * * F 1.30 1.52 Arg 85 A. . . . T . 0.92 * * F 1.00 1.12 Leu 86 A . . . . T . 0.97 . * . 0.851.13 Arg 87 A . . B . . . 1.24 . * . 0.75 1.46 Val 88 A . . B . . .0.84 * * . 0.75 1.08 His 89 A . . B . . . 1.10 . * . −0.15 1.13 Lys 90 A. . B . . . 0.29 * * F 0.90 1.16 Thr 91 . . B B . . . 0.24 * * F 0.001.35 Phe 92 . . B B . . . −0.72 * * . −0.30 0.74 Lys 93 . . B B . . .−0.72 * * . −0.30 0.27 Phe 94 . . B B . . . −1.03 * . . −0.60 0.14 Val95 . . B B . . . −1.93 * . . −0.60 0.16 Val 96 . . B B . . . −2.43 . * .−0.60 0.06 Val 97 . . B B . . . −2.54 . * . −0.60 0.06 Gly 98 . . B B .. . −2.59 . * . −0.60 0.06 Val 99 . . B B . . . −2.74 . . . −0.60 0.15Leu 100 . . B B . . . −2.74 * . . −0.60 0.15 Leu 101 . . B B . . .−2.10 * . . −0.60 0.11 Gln 102 . . B B . . . −1.54 * . . −0.60 0.23 Val103 . . B B . . . −1.50 . . . −0.60 0.37 Val 104 . . B . . T . −1.23 . .. −0.20 0.61 Pro 105 . . B . . T . −1.01 * . F 0.25 0.35 Ser 106 A . . .. T . −0.51 * . F −0.05 0.48 Ser 107 A . . . . T . −1.40 * * F 0.25 0.94Ala 108 A . . . . . . −0.50 . * F 0.05 0.43 Ala 109 A . . . . . . −0.46. * . 0.50 0.63 Thr 110 A . . . . . . −0.28 . * . −0.10 0.39 Ile 111 A .. . . . . 0.02 . * . −0.10 0.53 Lys 112 . . B . . . . 0.32 . * . 0.500.87 Leu 113 . . B . . . . 0.61 . * F 1.05 1.04 His 114 . . B . . . .0.31 . * F 1.30 1.99 Asp 115 . . . . . T C 0.28 * * F 1.80 0.70 Gln 116. . . . T T . 0.86 . * F 1.65 0.84 Ser 117 . . . . T T . 0.81 . . F 2.500.89 Ile 118 . . . . T T . 1.62 . . F 2.25 0.92 Gly 119 . . . . . . C1.37 . . F 1.00 0.92 Thr 120 . . . . . . C 1.37 . . F 0.45 0.72 Gln 121. . B . . . C 1.33 . . F 0.65 1.79 Gln 122 . . B . . . . 1.33 . . F 0.202.46 Trp 123 . . B . . . . 2.01 . . . 0.05 2.28 Glu 124 . . . . . . C1.54 . . . 0.25 2.04 His 125 . . . . . . C 1.51 . . . 0.10 0.97 Ser 126. . . . . T C 1.51 . . F 0.45 0.91 Pro 127 . . . . T T . 0.70 . . F 1.550.91 Leu 128 . . . . T T . 0.32 . . F 0.65 0.55 Gly 129 . . . . T T .0.11 . . F 0.65 0.22 Glu 130 . . . . T . . −0.07 . . F 0.45 0.22 Leu 131. . B . . . . −0.11 * . . 0.18 0.42 Cys 132 . . B . . . . −0.20 * . F1.21 0.42 Pro 133 . . B . . T . 0.58 * * F 1.69 0.32 Pro 134 . . . . T T. 1.03 . * F 1.47 0.53 Gly 135 . . . . T T . 0.73 . * F 2.80 1.94 Ser136 . . . . . T C 1.54 * . F 2.32 1.68 His 137 . . . . . . C 2.32 * . F2.48 1.88 Arg 138 . . B . . . . 2.32 * . F 2.34 3.72 Ser 139 . . B . . .. 2.19 * . F 2.40 4.29 Glu 140 . . . . T . . 1.94 * . F 2.86 3.12 Arg141 . . . . T T . 1.58 * . F 3.40 1.61 Pro 142 . . . . T T . 1.61 . * F2.91 0.64 Gly 143 . . . . T T . 1.61 . * F 2.57 0.60 Ala 144 . . . . T T. 1.24 . * . 2.08 0.60 Cys 145 . . . . T . . 0.93 . * . 1.41 0.21 Asn146 . . B . . . . 0.82 . * . 0.84 0.30 Arg 147 . . B . . . . 0.69 * . .1.01 0.52 Cys 148 . . B . . T . 0.18 * . F 1.83 0.96 Thr 149 . . B . . T. 0.42 * . F 1.70 0.44 Glu 150 . . B . . T . 0.84 * . F 1.53 0.22 Gly151 . . B . . T . 0.53 * . F 0.76 0.65 Val 152 . . B B . . . 0.42 . * F0.19 0.65 Gly 153 . . B B . . . 0.50 . . . −0.13 0.61 Tyr 154 . . B B .. . 0.51 . . . −0.60 0.62 Thr 155 . . B B . . . 0.51 . . F −0.30 1.12Asn 156 . . . B . . C 0.86 . . F 0.20 1.81 Ala 157 . . . . T T . 0.90 .. F 0.80 1.86 Ser 158 . . . . T T . 0.54 . . F 0.80 1.06 Asn 159 . . . .T T . 0.20 . . F 0.35 0.57 Asn 160 . . . . T T . −0.16 * . F 0.35 0.57Leu 161 . A B . . . . −0.97 * . . −0.60 0.23 Phe 162 . A B . . . . −0.59. . . −0.60 0.12 Ala 163 . A B . . . . −0.96 . . . −0.60 0.11 Cys 164 .A B . . . . −1.27 * . . −0.60 0.07 Leu 165 . . B . . T . −1.86 . . .−0.20 0.12 Pro 166 . . B . . T . −1.71 * . . −0.20 0.12 Cys 167 . . . .T T . −0.97 * . . 0.20 0.12 Thr 168 A . . . . T . −0.68 . . . 0.10 0.30Ala 169 A . . . . . . −0.01 . . . 0.50 0.26 Cys 170 A . . . . T . 0.80 .. . 0.70 0.80 Lys 171 A . . . . T . 1.01 . . F 1.15 0.96 Ser 172 A . . .. T . 1.68 . * F 1.30 1.65 Asp 173 A . . . . T . 2.10 . * F 1.30 5.33Glu 174 A A . . . . . 2.39 . * F 0.90 5.22 Glu 175 A A . . . . . 2.84. * F 1.24 5.22 Glu 176 A A . . . . . 2.13 . * F 1.58 4.83 Arg 177 . A .. T . . 2.12 . . F 2.32 1.50 Ser 178 . . . . . T C 1.81 . . F 2.86 1.25Pro 179 . . . . T T . 1.50 * . F 3.40 1.04 Cys 180 . . . . T T . 1.61 *. F 2.61 0.77 Thr 181 . . . . T T . 1.61 * . F 2.67 1.12 Thr 182 . . . .T . . 1.19 * * F 2.38 1.16 Thr 183 . . . . T T . 0.90 . . F 2.49 3.13Arg 184 . . . . T T . 0.44 . . F 2.40 2.19 Asn 185 . . . . T T . 1.11 .. F 2.50 0.81 Thr 186 . . . . T T . 0.76 * . F 2.25 0.98 Ala 187 . . . .T . . 1.11 * . . 1.65 0.27 Cys 188 . . . . T . . 1.21 * . . 1.40 0.33Gln 189 . . B . . . . 0.76 * . . 0.75 0.36 Cys 190 . . B . . . . 0.44 .. . 0.50 0.35 Lys 191 . . B . . T . 0.06 . * F 0.85 0.94 Pro 192 . . . .T T . 0.76 . . F 0.65 0.47 Gly 193 . . . . T T . 1.42 . * F 1.74 1.72Thr 194 . . B . . T . 1.42 . * F 1.68 1.38 Phe 195 . . B . . . . 2.09. * F 1.82 1.49 Arg 196 . . . . T . . 1.74 . * F 2.56 2.42 Asn 197 . . .. T T . 1.37 . * F 3.40 2.25 Asp 198 . . . . T T . 1.71 . * F 3.06 2.63Asn 199 . . . . . T C 1.42 . * F 2.52 2.32 Ser 200 A . . . . T . 1.46. * F 1.98 1.43 Ala 201 A . . . . . . 1.46 . * . 1.14 0.46 Glu 202 A . .. . . . 1.50 * . . 0.80 0.56 Met 203 A . . . . . . 0.83 * . . 1.11 0.83Cys 204 A . . . . T . 0.53 * . . 1.62 0.44 Arg 205 . . . . T T . 0.52 *. . 2.33 0.34 Lys 206 . . . . T T . 0.77 * . F 2.49 0.50 Cys 207 . . . .T T . 0.10 * . F 3.10 0.92 Ser 208 . . . . T . . 0.49 * * F 2.59 0.25Thr 209 . . . . T . . 1.27 * * F 1.98 0.19 Gly 210 . . . . T . . 0.81 *. F 1.67 0.71 Cys 211 . . B . . T . 0.17 * * F 1.16 0.53 Pro 212 . . . .T T . −0.02 * * F 1.25 0.36 Arg 213 . . . . T T . 0.32 * * F 0.65 0.27Gly 214 . . B . . T . −0.22 * * . 0.85 1.01 Met 215 . . B B . . .0.17 * * . 0.30 0.48 Val 216 . . B B . . . 0.83 * * . 0.79 0.49 Lys 217. . B B . . . 0.38 * * . 0.98 0.83 Val 218 . . B B . . . −0.04 * * F1.32 0.45 Lys 219 . . B B . . . 0.09 . * F 1.51 0.88 Asp 220 . . B . . .. 0.40 . * F 1.90 0.68 Cys 221 . . B . . . . 0.96 . * F 0.81 0.96 Thr222 . . . . . T C 0.91 . * F 1.62 0.65 Pro 223 . . . . T T . 0.88 . * F1.63 0.65 Trp 224 . . . . T T . 0.83 . * F 0.54 0.84 Ser 225 A . . . . T. 0.17 . . F 1.00 1.01 Asp 226 A A . . . . . −0.02 . . F 0.45 0.35 Ile227 A A . . . . . 0.26 * . . −0.30 0.25 Glu 228 A A . . . . . 0.51 * . .0.30 0.25 Cys 229 . A B . . . . 0.80 * . . 0.60 0.30 Val 230 A A . . . .. 0.80 * * . 0.60 0.74 His 231 A A . . . . . 0.46 * * . 0.60 0.58 Lys232 A A . . . . . 1.34 * . F 0.60 1.06 Glu 233 . A . . T . . 1.00 * . F1.30 2.30 Ser 234 . . . . T T . 1.63 * . F 1.70 1.68 Gly 235 . . . . T T. 2.49 * . F 1.70 1.14 Asn 236 . . . . T T . 1.63 * . F 1.40 1.06 Gly237 . . . . . T C 1.30 * . F 0.45 0.55 His 238 . . . B . . C 0.44 . . .−0.40 0.59 Asn 239 . . . B . . C −0.14 . . . −0.40 0.27 Ile 240 . . B B. . . −0.61 . . . −0.60 0.19 Trp 241 . . B B . . . −1.47 . . . −0.600.12 Val 242 . . B B . . . −1.98 . . . −0.60 0.05 Ile 243 . . B B . . .−2.26 . . . −0.60 0.06 Leu 244 . . B B . . . −3.07 . . . −0.60 0.08 Val245 . . B B . . . −3.03 . . . −0.60 0.09 Val 246 . . B B . . . −3.60 . .. −0.60 0.09 Thr 247 . . B B . . . −2.96 . . . −0.60 0.08 Leu 248 . . BB . . . −2.88 . . . −0.60 0.17 Val 249 . . B B . . . −2.88 . * . −0.600.19 Val 250 . . B B . . . −2.83 . . . −0.60 0.11 Pro 251 . . B B . . .−2.83 . . . −0.60 0.11 Leu 252 . . B B . . . −3.11 . . . −0.60 0.11 Leu253 A . . B . . . −3.16 . . . −0.60 0.15 Leu 254 A . . B . . . −3.11 . .. −0.60 0.07 Val 255 A . . B . . . −3.14 . . . −0.60 0.07 Ala 256 A . .B . . . −3.79 . . . −0.60 0.06 Val 257 . . B B . . . −3.64 . . . −0.600.05 Leu 258 . . B B . . . −3.50 . . . −0.60 0.04 Ile 259 . . B B . . .−3.36 . . . −0.60 0.02 Val 260 . . B B . . . −3.39 . . . −0.60 0.02 Cys261 . . B B . . . −3.14 . . . −0.60 0.01 Cys 262 . . B B . . . −2.59 . .. −0.60 0.02 Cys 263 . . B B . . . −2.12 . . . −0.60 0.03 Ile 264 . . BB . . . −1.90 . . . −0.60 0.06 Gly 265 . . . . T T . −1.39 . . F 0.350.06 Ser 266 . . . . T T . −1.07 . . F 0.35 0.11 Gly 267 . . . . T T .−0.40 . . F 0.65 0.16 Cys 268 . . . . T T . 0.06 . . F 1.25 0.27 Gly 269. . . . T . . 0.99 . * F 1.39 0.31 Gly 270 . . . . T . . 0.67 . . F 2.030.62 Asp 271 . . . . . T C 0.37 . . F 2.37 0.62 Pro 272 . . . . T T .0.71 * * F 2.91 0.62 Lys 273 . . . . T T . 1.49 * * F 3.40 1.05 Cys 274. . B . . T . 0.98 * * . 2.51 1.23 Met 275 . . B B . . . 0.66 * * . 1.620.59 Asp 276 . . B B . . . −0.04 * * . 1.28 0.16 Arg 277 . . B B . . .−0.12 . * . 0.04 0.26 Val 278 . . B B . . . −0.06 . * . −0.60 0.27 Cys279 . . B B . . . −0.20 . . . 0.30 0.32 Phe 280 . . B B . . . 0.06 . * .−0.60 0.13 Trp 281 . . B B . . . −0.76 . . . −0.60 0.18 Arg 282 . . B B. . . −1.68 . . . −0.60 0.28 Leu 283 . . B B . . . −0.71 . . . −0.600.26 Gly 284 . . . B T . . −0.39 . * . −0.20 0.49 Leu 285 . . . B . . C0.10 . * . 0.50 0.25 Leu 286 . . . B . . C 0.04 . * . 0.20 0.46 Arg 287. . . B . . C −0.66 . . F 0.65 0.46 Gly 288 . . . . . T C 0.16 . . F1.35 0.57 Pro 289 . . . . . T C 0.50 . * F 2.70 1.19 Gly 290 . . . . . TC 1.31 * * F 3.00 1.01 Ala 291 A . . . . T . 1.53 . * F 2.50 1.65 Glu292 A . . . . . . 1.39 . . F 2.00 1.08 Asp 293 A . . . . . . 1.73 . . F1.70 1.48 Asn 294 A . . . . T . 1.94 . * . 1.45 2.36 Ala 295 A . . . . T. 1.40 . . . 1.15 2.36 His 296 A . . . . T . 1.18 * . . 1.00 0.99 Asn297 A . . . . T . 0.88 . . . 0.10 0.51 Glu 298 A . . . . . . 0.88 * . .−0.10 0.67 Ile 299 A . . . . . . 0.29 * * . −0.10 0.80 Leu 300 A . . . .. . 0.88 * * . −0.10 0.50 Ser 301 A . . . . . . 0.61 * . F 0.65 0.48 Asn302 A . . . . T . −0.20 * . F 0.25 0.92 Ala 303 A . . . . T . −0.50 * .F 0.25 0.92 Asp 304 A . . . . T . 0.08 * . F 0.85 0.92 Ser 305 . . . . .T C 0.19 * . F 1.05 0.83 Leu 306 . . . B . . C −0.37 * . F 0.05 0.71 Ser307 . . B B . . . −0.67 * . F −0.15 0.31 Thr 308 . . B B . . . −0.08 * .. −0.60 0.31 Phe 309 . . B B . . . −0.08 * . . −0.30 0.66 Val 310 A . .B . . . 0.22 . . F −0.15 0.85 Ser 311 A A . . . . . 0.43 . . F 0.00 1.03Glu 312 A A . . . . . 0.73 . . F 0.00 1.17 Gln 313 A A . . . . . 0.74 .. F 0.90 2.73 Gln 314 A A . . . . . 1.44 . . F 0.90 2.73 Met 315 A A . .. . . 2.30 . . F 0.90 2.73 Glu 316 A A . . . . . 2.39 . . F 0.90 2.73Ser 317 A A . . . . . 1.80 . * F 0.90 2.44 Gln 318 A A . . . . . 1.80. * F 0.90 2.49 Glu 319 A A . . . . . 0.99 . * F 0.90 2.40 Pro 320 A A .. . . . 1.28 . * F 0.90 1.48 Ala 321 A A . . . . . 0.93 . . F 0.60 1.23Asp 322 A A . B . . . 0.38 . . F 0.45 0.70 Leu 323 A A . B . . . 0.07 .. F −0.15 0.34 Thr 324 . A B B . . . −0.79 . . F −0.15 0.48 Gly 325 . AB B . . . −0.58 . . . −0.30 0.21 Val 326 . . B B . . . −0.29 . . . −0.600.45 Thr 327 . . B B . . . −0.50 . . . −0.60 0.42 Val 328 . . B B . . .−0.03 . * F −0.17 0.65 Gln 329 . . B B . . . 0.28 . * F 0.11 0.87 Ser330 . . . . . T C 0.03 . * F 2.04 1.05 Pro 331 . . . . . T C 0.89 . * F2.32 1.42 Gly 332 . . . . T T . 0.53 . * F 2.80 1.42 Glu 333 A . . . . T. 0.58 . * F 1.97 0.57 Ala 334 . . B . . . . −0.23 . * . 0.74 0.30 Gln335 . . B . . . . −0.28 . . . 0.46 0.25 Cys 336 . . B . . . . −0.28 . .. 0.18 0.14 Leu 337 . . B . . . . −0.52 . * . −0.40 0.22 Leu 338 . . B .. . . −0.52 . * . −0.40 0.13 Gly 339 . A . . . . C −0.52 . * F 0.05 0.42Pro 340 A A . . . . . −0.52 . * F −0.15 0.51 Ala 341 A A . . . . . −0.20. * F 0.60 1.07 Glu 342 A A . . . . . 0.31 . * F 0.90 1.07 Ala 343 A A .. . . . 1.12 * * F 0.75 0.93 Glu 344 A A . . . . . 1.58 . * F 0.90 1.60Gly 345 A A . . . . . 1.90 . * F 0.90 1.80 Ser 346 A . . . . T . 2.60. * F 1.30 3.50 Gln 347 A . . . . T . 1.79 . * F 1.30 3.96 Arg 348 A . .. . T . 1.57 . * F 1.30 3.30 Arg 349 . . B . . T . 0.71 . * F 1.30 2.03Arg 350 . . B B . . . 0.84 . * F 0.75 0.87 Leu 351 . . B B . . . 0.56. * . 0.60 0.69 Leu 352 . . B B . . . 0.56 . * . 0.30 0.35 Val 353 . . BB . . . 0.10 * * . −0.30 0.29 Pro 354 . . B . . T . −0.60 * . . −0.200.35 Ala 355 . . . . T T . −0.71 . * . 0.50 0.43 Asn 356 . . . . . T C−0.11 . . F 1.65 0.96 Gly 357 . . . . . T C 0.39 . . F 1.95 0.96 Ala 358. . . . . . C 1.24 . . F 2.20 1.37 Asp 359 . . . . . T C 1.14 . . F 3.001.48 Pro 360 A . . . . T . 0.92 * . F 2.50 2.16 Thr 361 A . . . . T .0.32 . . F 1.90 1.76 Glu 362 A . . . . T . −0.14 . . F 1.60 1.04 Thr 363A . . B . . . −0.26 . . F 0.15 0.56 Leu 364 A . . B . . . −0.96 * . .−0.60 0.33 Met 365 A . . B . . . −0.74 * . . −0.60 0.17 Leu 366 A . . B. . . −0.39 * . . −0.60 0.19 Phe 367 A . . B . . . −1.09 * . . −0.600.47 Phe 368 A . . B . . . −1.37 * . . −0.60 0.41 Asp 369 A . . B . . .−0.56 * . . −0.60 0.50 Lys 370 A A . . . . . −0.84 * . . −0.30 0.93 Phe371 A A . B . . . −0.89 * . . −0.30 0.75 Ala 372 A A . B . . . −0.40 * .. −0.30 0.34 Asn 373 . A B B . . . −0.40 * . . −0.60 0.26 Ile 374 . A BB . . . −0.40 * . . −0.60 0.26 Val 375 . A B B . . . −0.74 . . . −0.600.43 Pro 376 . A . B . . C −0.33 . . . −0.10 0.36 Phe 377 . . . . T T .0.26 . . . 0.20 0.54 Asp 378 . . . . T T . 0.26 . . F 0.80 1.21 Ser 379. . . . T T . 0.33 . . F 1.40 1.35 Trp 380 A . . . . T . 0.59 * * F 0.401.29 Asp 381 A A . . . . . 0.91 * . F −0.15 0.76 Gln 382 A A . . . . .1.61 * . . −0.15 1.11 Leu 383 A A . . . . . 0.80 * . . −0.15 1.84 Met384 A A . . . . . 1.10 * . . 0.30 0.91 Arg 385 A A . . . . . 0.58 * . .0.30 0.87 Gln 386 A A . . . . . 0.27 * . . −0.30 0.87 Leu 387 A A . . .. . 0.31 * . . 0.45 1.27 Asp 388 A A . . . . . 1.12 * . . 0.75 1.30 Leu389 A A . . . . . 1.72 * . F 0.60 1.21 Thr 390 A . . . . T . 0.72 * . F1.30 2.54 Lys 391 A . . . . T . 0.72 . * F 1.30 1.07 Asn 392 A . . . . T. 0.68 * * F 1.30 2.16 Glu 393 A . . . . T . −0.18 * . F 1.30 1.11 Ile394 . . B B . . . 0.74 * . F 0.75 0.41 Asp 395 . . B B . . . 0.47 * * .0.60 0.50 Val 396 . . B B . . . 0.08 * * . 0.60 0.29 Val 397 . . B B . .. −0.23 . . . 0.51 0.41 Arg 398 . . B . . T . −0.82 * . . 1.12 0.36 Ala399 . . B . . T . −0.28 * . . 0.73 0.49 Gly 400 . . . . T T . −0.49 * .F 2.09 0.65 Thr 401 . . . . . T C 0.02 * * F 2.10 0.51 Ala 402 . . . . .. C 0.88 * * F 1.09 0.50 Gly 403 . . . . . T C 0.18 * * F 1.68 0.85 Pro404 . . . . . T C −0.04 . . F 1.47 0.59 Gly 405 . . . . . T C 0.06 . . F1.26 0.48 Asp 406 A . . . . T . −0.22 . . F 0.25 0.76 Ala 407 A A . . .. . −0.23 . . . −0.30 0.50 Leu 408 A A . . . . . −0.70 . . . −0.60 0.50Tyr 409 A A . . . . . −1.09 * . . −0.60 0.25 Ala 410 A A . . . . .−0.70 * . . −0.60 0.24 Met 411 A A . . . . . −0.99 * . . −0.60 0.59 Leu412 A A . . . . . −1.26 * . . −0.60 0.39 Met 413 A A . . . . . −0.44 * .. −0.60 0.29 Lys 414 A A . B . . . −0.16 * . . −0.60 0.47 Trp 415 A A .B . . . 0.12 * . . 0.15 1.14 Val 416 A A . B . . . 0.38 * * . 0.45 1.66Asn 417 A . . . . T . 1.30 * . F 1.75 0.82 Lys 418 A . . . . T . 1.90 *. F 2.20 1.53 Thr 419 . . . . . T C 1.27 * . F 3.00 3.32 Gly 420 . . . .. T C 1.26 * . F 2.70 2.08 Arg 421 . . . . T . . 1.22 * . F 2.40 1.40Asn 422 . . . . . T C 1.19 * . F 1.65 0.68 Ala 423 . . B . . T . 0.83 .. . 1.00 0.93 Ser 424 . . B . . T . 0.33 . . . 0.70 0.69 Ile 425 . . B .. T . −0.13 . * . −0.20 0.35 His 426 . A B . . . . −0.24 . * . −0.600.29 Thr 427 . A B . . . . −0.83 * * . −0.60 0.36 Leu 428 A A . . . . .−1.06 * * . −0.60 0.52 Leu 429 A A . . . . . −0.76 * * . −0.60 0.31 Asp430 A A . . . . . 0.24 * * . −0.30 0.38 Ala 431 A A . . . . . −0.32 * *. 0.30 0.89 Leu 432 A A . . . . . −0.01 * * . 0.75 1.07 Glu 433 A A . .. . . 0.80 * * . 0.75 1.11 Arg 434 A A . . . . . 1.72 * * F 0.90 1.90Met 435 A A . . . . . 1.69 * * F 0.90 4.52 Glu 436 A A . . . . .1.69 * * F 0.90 3.55 Glu 437 A A . . . . . 2.54 * . F 0.90 1.83 Arg 438A A . . . . . 2.54 * * F 0.90 3.70 His 439 A A . . . . . 2.48 * * F 0.903.70 Ala 440 A A . . . . . 2.19 * * F 0.90 4.28 Lys 441 A A . . . . .2.19 * * F 0.90 1.53 Glu 442 A A . . . . . 2.19 * . F 0.90 1.95 Lys 443A A . . . . . 1.27 * * F 0.90 3.22 Ile 444 A A . . . . . 0.49 * * F 0.901.33 Gln 445 A A . . . . . 0.22 * * F 0.75 0.63 Asp 446 A A . . . . .0.18 * * F −0.15 0.23 Leu 447 A A . . . . . −0.12 * . . −0.30 0.56 Leu448 A A . . . . . −0.51 * . . 0.55 0.43 Val 449 A A . . . . . 0.42 * . F0.95 0.26 Asp 450 A . . . . T . −0.28 * . F 1.60 0.62 Ser 451 . . . . TT . −1.17 * . F 2.25 0.65 Gly 452 . . . . T T . −0.60 * . F 2.50 0.62Lys 453 . . B . . T . −0.60 . . F 1.25 0.58 Phe 454 . A B . . . . 0.26 .. . 0.15 0.36 Ile 455 . A B . . . . 0.26 . . . 0.20 0.62 Tyr 456 . A B .. . . 0.21 . . . 0.55 0.52 Leu 457 . A B . . . . 0.24 . . . −0.03 0.59Glu 458 . A B . . . . −0.14 . . F 0.54 1.22 Asp 459 . A . . T . . 0.26 .. F 1.66 0.77 Gly 460 . . . . T T . 0.56 . . F 2.78 1.26 Thr 461 . . . .. T C −0.06 * . F 2.70 0.73 Gly 462 . . . . . T C 0.46 * . F 2.13 0.33Ser 463 . . . . . T C −0.36 . . F 1.26 0.44 Ala 464 A . . . . . . −0.36. . . 0.14 0.25 Val 465 . . B . . . . −0.40 . . . 0.17 0.44 Ser 466 . .B . . . . −0.48 . . . −0.10 0.42 Leu 467 . . B . . . . −0.52 . . . −0.100.53 Glu 468 A . . . . . . −0.61 . . . 0.50 0.92

Preferred nucleic acid fragments of the invention encode a full-lengthDR4 polypeptide lacking the nucleotides encoding the amino-terminalmethionine (nucleotides 19-21 in SEQ ID NO:1) as it is known that themethionine is cleaved naturally and such sequences maybe useful ingenetically engineering DR4 expression vectors. Polypeptides encoded bysuch polynucleotides are also contemplated by the invention.

Among highly preferred fragments in this regard are those that comprise,or alternatively consist of, regions of DR4 that combine severalstructural features, such as several of the features set out above.Preferred nucleic acid fragments of the present invention furtherinclude nucleic acid molecules encoding a polypeptide comprising, oralternatively consisting of, one, two, three, four, five, or moreepitope-bearing portions of the DR4 protein. In particular, such nucleicacid fragments of the present invention include nucleic acid moleculesencoding: a polypeptide comprising, or alternatively consisting of,amino acid residues from about 35 to about 92 in SEQ ID NO:2; apolypeptide comprising, or alternatively consisting of, amino acidresidues from about 114 to about 160 in SEQ ID NO:2; a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 169 to about 240 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 267 to about298 in SEQ ID NO:2; a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 330 to about 364 in SEQ IDNO:2; a polypeptide comprising, or alternatively consisting of, aminoacid residues from about 391 to about 404 in SEQ ID NO:2; and apolypeptide comprising, or alternatively consisting of, amino acidresidues from about 418 to about 465 in SEQ ID NO:2. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) amino acid residues, at either terminus or atboth termini. The inventors have determined that the above polypeptidefragments are antigenic regions of the DR4 protein. Methods fordetermining other such epitope-bearing portions of the DR4 protein aredescribed in detail below. Polypeptides encoded by these nucleic acidsare also encompassed by the invention.

In specific embodiments, the polynucleotides of the invention are lessthan 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb,30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.

In further embodiments, polynucleotides of the invention comprise, oralternatively consist of, at least 15, at least 30, at least 50, atleast 100, or at least 250, at least 500, or at least 1000 contiguousnucleotides of DR4 coding sequence, but consist of less than or equal to1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5′ or 3′coding nucleotide set forth in SEQ ID NO: 1. In further embodiments,polynucleotides of the invention comprise, or alternatively consist of,at least 15, at least 30, at least 50, at least 100, or at least 250, atleast 500, or at least 1000 contiguous nucleotides of DR4 codingsequence, but do not comprise all or a portion of any DR4 intron. Inanother embodiment, the nucleic acid comprising, or alternativelyconsisting of, DR4 coding sequence does not contain coding sequences ofa genomic flanking gene (i.e., 5′ or 3′ to the DR4 gene in the genome).In other embodiments, the polynucleotides of the invention do notcontain the coding sequence of more than 1000, 500, 250, 100, 50, 25,20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 asfollows: HTOIY07R (SEQ ID NO:6) and HTXEY80R (SEQ ID NO:7) both shown inFIG. 4.

Further, the invention includes a polynucleotide comprising, oralternatively consisting of, any portion of at least about 30nucleotides, preferably at least about 50 nucleotides, of SEQ ID NO:1from residue 365 to 1,422. In this context “about” includes theparticularly recited size, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidewhich hybridizes under stringent hybridization conditions to a portionof the polynucleotide in a nucleic acid molecule of the inventiondescribed above, for instance, the sequence complementary to the codingand/or noncoding (i.e., transcribed, untranslated) sequence depicted inSEQ ID NO:1, the cDNA contained in ATCC Deposit No. 97853, and thesequence encoding a DR4 domain or a polynucleotide fragment as describedherein. By “stringent hybridization conditions” is intended overnightincubation at 42° C. in a solution comprising, or alternativelyconsisting of: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Polypeptidesencoded by these nucleic acids are also encompassed by the invention.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. In this context “about”includes the particularly recited size, larger or smaller by several (5,4, 3, 2, or 1) nucleotides, at either terminus or at both termini. By aportion of a polynucleotide of “at least 20 nt in length,” for example,is intended 20 or more contiguous nucleotides from the nucleotidesequence of the reference polynucleotide (e.g., the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:1. These have uses, whichinclude, but are not limited to, as diagnostic probes and primers asdiscussed above and in more detail below.

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3 terminal poly(A) tract of the DR4 cDNA shown in SEQ IDNO:1), or to a complementary stretch of T (or U) resides, would not beincluded in a polynucleotide of the invention used to hybridize to aportion of a nucleic acid of the invention, since such a polynucleotidewould hybridize to any nucleic acid molecule containing a poly (A)stretch or the complement thereof (e.g., practically any double-strandedcDNA).

As indicated, nucleic acid molecules of the present invention whichencode the DR4 polypeptide may include, but are not limited to thecoding sequence for the mature polypeptide, by itself; the codingsequence for the mature polypeptide and additional sequences, such asthose encoding a leader or secretary sequence, such as a pre-, or pro-or prepro-protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag corresponds to an epitopederived of influenza hemagglutinin protein, which has been described byWilson et al., Cell 37:767 (1984), for instance.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode for fragments, analogsor derivatives of the DR4 polypeptide. Variants may occur naturally,such as an allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions which may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and functional activitiesof the DR4 receptor or portions thereof. Also especially preferred inthis regard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acidmolecules that are at least 80% identical, and more preferably at least85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical, to (a) a nucleotidesequence encoding the full-length DR4 polypeptide having the completeamino acid sequence in SEQ ID NO:2, including the predicted leadersequence; (b) nucleotide sequence encoding the full-length DR4polypeptide having the complete amino acid sequence in SEQ ID NO:2,including the predicted leader sequence but lacking the amino terminalmethionine; (c) a nucleotide sequence encoding the mature DR4polypeptide (full-length polypeptide with the leader removed) having theamino acid sequence at positions about 24 to about 468 in SEQ ID NO:2;(d) a nucleotide sequence encoding the full-length DR4 polypeptidehaving the complete amino acid sequence including the leader encoded bythe cDNA contained in ATCC Deposit No. 97853; (e) a nucleotide sequenceencoding the full-length DR4 polypeptide having the complete amino acidsequence including the leader but lacking the amino terminal methionineencoded by the cDNA contained in ATCC Deposit No. 97853; (f) anucleotide sequence encoding the mature DR4 polypeptide having the aminoacid sequence encoded by the cDNA contained in ATCC Deposit No. 97853;(g) a nucleotide sequence that encodes the DR4 extracellular domainhaving the amino acid sequence at positions about 24 to about 238 in SEQID NO:2, or the DR4 extracellular domain encoded by the cDNA containedin ATCC Deposit No. 97853; (h) a nucleotide sequence that encodes theDR4 transmembrane domain having the amino acid sequence at positionsabout 239 to about 264 in SEQ ID NO:2, or the DR4 transmembrane domainencoded by the cDNA contained in ATCC Deposit No. 97853; (i) anucleotide sequence that encodes the DR4 intracellular domain having theamino acid sequence at positions about 265 to about 468 in SEQ ID NO:2,or the DR4 intracellular domain encoded by the cDNA contained in ATCCDeposit No. 97853; (j) a nucleotide sequence that encodes the DR4 deathdomain having the amino acid sequence at positions about 379 to about422 in SEQ ID NO:2, or the DR4 death domain encoded by the cDNAcontained in ATCC Deposit No. 97853; (k) a nucleotide sequence thatencodes the DR4 cysteine rich domain having the amino acid sequence atpositions about 131 to about 229 in SEQ ID NO:2, or the DR4 cysteinerich domain encoded by the cDNA contained in ATCC Deposit No. 97853; (l)a nucleotide sequence that encodes the DR4 receptor extracellular andintracellular domains with all or part of the transmembrane domaindeleted; (m) a nucleotide sequence that encodes a fragment of thepolypeptide of (c) having DR4 functional activity (e.g., antigenic orbiological activity); or (n) a nucleotide sequence complementary to anyof the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h),(i), (j), (k), (l), or (m) above. In this context “about” includes theparticularly recited size, larger or smaller by several (5, 4, 3, 2,or 1) amino acid residues, at either terminus or at both termini.Polypeptides encoded by these nucleic acids are also encompassed by theinvention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a DR4polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five mismatches per each 100nucleotides of the reference nucleotide sequence encoding the DR4polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mismatches of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entire DR4nucleotide sequence shown in SEQ ID NO:1 or any fragment (e.g., apolynucleotide encoding the amino acid sequence of a DR4 N- and/orC-terminal deletion described herein) as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in SEQ ID NO:1 or to thenucleotide sequences of the deposited cDNA can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

The present application is directed to nucleic acid molecules at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleicacid sequence shown in SEQ ID NO:1 or to the nucleic acid sequence ofthe deposited cDNAs, irrespective of whether they encode a polypeptidehaving DR4 functional activity. This is because even where a particularnucleic acid molecule does not encode a polypeptide having DR4functional activity, one of skill in the art would still know how to usethe nucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving DR4 functional activity include, inter alia, (1) isolating theDR4 gene or allelic variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the DR4 gene, as described in Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988); and (3) Northern Blot analysis for detecting DR4 mRNAexpression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleicacid sequence shown in SEQ ID NO:1 or to the nucleic acid sequence ofthe deposited cDNAs which do, in fact, encode a polypeptide having DR4protein functional activity. By “a polypeptide having DR4 proteinfunctional activity” is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to an activity of the DR4protein of the invention (either the full-length protein (i.e. complete)or, preferably, the mature protein), as measured in a particularfunctional and/or biological assay. For example, DR4 polypeptidefunctional activity can be measured by the ability of a polypeptidesequence described herein to form multimers (e.g., homodimers andhomotrimers) with complete DR4, and to bind a DR4 ligand (e.g., TRAIL).These functional assays can be routinely performed using techniquesdescribed herein and otherwise known in the art.

For example, DR4 protein functional activity (e.g., biological activity)can routinely be measured using the cell death assays performedessentially as previously described (A. M. Chinnaiyan, et al., Cell 81,505-12 (1995); M. P. Boldin, et al., J Biol Chem 270, 7795-8 (1995); F.C. Kischkel, et al., EMBO 14, 5579-5588 (1995); A. M. Chinnaiyan, etal., J Biol Chem 271, 4961-4965 (1996)) or as set forth in Example 5,below. In MCF7 cells, plasmids encoding full-length DR4 or a candidatedeath domain containing receptors are co-transfected with the pLanternreporter construct encoding green fluorescent protein. Nuclei of cellstransfected with DR4 will exhibit apoptotic morphology as assessed byDAPI staining. Similar to TNFR-1 and Fas/APO-1 (M. Muzio, et al., Cell85, 817-827 (1996); M. P. Boldin, et al., Cell 85, 803-815 (1996); M.Tewari, et al., J Biol Chem 270, 3255-60 (1995)), DR4-induced apoptosisis blocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98% or 99% identical to, for example, the nucleic acidsequence of the deposited cDNA or the nucleic acid sequence shown in SEQID NO:1 will encode “a polypeptide having DR4 protein functionalactivity.” In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, this will be clear to theskilled artisan even without performing the above described comparisonassay. It will be further recognized in the art that, for such nucleicacid molecules that are not degenerate variants, a reasonable numberwill also encode a polypeptide having DR4 protein functional activity.This is because the skilled artisan is fully aware of amino acidsubstitutions that are either less likely or not likely to significantlyeffect protein function (e.g., replacing one aliphatic amino acid with asecond aliphatic amino acid), as further described below.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Polynucleotide Assays

This invention is also related to the use of the DR4 polynucleotides todetect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of a mutated form of DR4 associated with adysfunction will provide a diagnostic tool that can add or define adiagnosis of a disease or susceptibility to a disease which results fromunder-expression over-expression or altered expression of DR4 or asoluble form thereof, such as, for example, tumors or autoimmunedisease.

Individuals carrying mutations in the DR4 gene may be detected at theDNA level by a variety of techniques. Nucleic acids for diagnosis may beobtained from a patient's cells, such as from blood, urine, saliva,tissue biopsy and autopsy material. The genomic DNA may be used directlyfor detection or may be amplified enzymatically by using PCR prior toanalysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA mayalso be used in the same ways. As an example, PCR primers complementaryto the nucleic acid encoding DR4 can be used to identify and analyze DR4expression and mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled DR4RNA or alternatively, radiolabeled DR4antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Vectors and Host Cells

The present invention also relates to vectors which include DNAmolecules of the present invention, host cells which are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express polypeptides of the present invention. Thepolynucleotides may be introduced alone or with other polynucleotides.Such other polynucleotides may be introduced independently,co-introduced or introduced joined to the polynucleotides of theinvention.

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well-knowntechniques for introducing DNA and RNA into cells. Viral vectors may bereplication competent or replication defective. In the latter case viralpropagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s)), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name just a few of the well-known promoters. In general,expression constructs will contain sites for transcription, initiationand termination, and, in the transcribed region, a ribosome binding sitefor translation. The coding portion of the mature transcripts expressedby the constructs will include a translation initiating AUG at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention.

Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. These vectors are listed solely by way of illustrationof the many commercially available and well known vectors available tothose of skill in the art.

Selection of appropriate vectors and promoters for expression in a hostcell is a well-known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

The present invention also relates to host cells containing theabove-described vector constructs described herein, and additionallyencompasses host cells containing nucleotide sequences of the inventionthat are operably associated with one or more heterologous controlregions (e.g., promoter and/or enhancer) using techniques known of inthe art. The host cell can be a higher eukaryotic cell, such as amammalian cell (e.g., a human derived cell), or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. The host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., phosphorylation, cleavage) of proteins. Appropriatecell lines can be chosen to ensure the desired modifications andprocessing of the foreign protein expressed.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods in MolecularBiology (1986).

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., DR4 coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with DR4 polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous DR4 polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous DR4 polynucleotide sequences via homologous recombination(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication Number WO 96/29411, published Sep. 26, 1996; InternationalPublication Number WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,Nature 342:435-438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Such a fusion protein can be made byligating polynucleotides of the invention and the desired nucleic acidsequence encoding the desired amino acid sequence to each other, bymethods known in the art, in the proper reading frame, and expressingthe fusion protein product by methods known in the art. Alternatively,such a fusion protein can be made by protein synthetic techniques, e.g.,by use of a peptide synthesizer. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. For example, in one embodiment,polynucleotides encoding DR4 polypeptides of the invention may be fusedto the pelB pectate lyase signal sequence to increase the efficiency toexpression and purification of such polypeptides in Gram-negativebacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents ofwhich are herein incorporated by reference in their entireties.

The addition of peptide moieties to polypeptides to engender secretionor excretion, to improve stability and to facilitate purification, amongothers, are familiar and routine techniques in the art. A preferredfusion protein comprises a heterologous region from immunoglobulin thatis useful to solubilize proteins. For example, EP-A-O 464 533 (Canadiancounterpart 2045869) discloses fusion proteins comprising variousportions of constant region of immunoglobin molecules together withanother human protein or part thereof. In many cases, the Fc part in afusion protein is thoroughly advantageous for use in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified in the advantageous mannerdescribed. This is the case when Fc portion proves to be a hindrance touse in therapy and diagnosis, for example when the fusion protein is tobe used as antigen for immunizations. In drug discovery, for example,human proteins, such as, hIL5-receptor has been fused with Fc portionsfor the purpose of high-throughput screening assays to identifyantagonists of hIL-5. See, D. Bennett et al., Journal of MolecularRecognition, Vol. 8:52-58 (1995) and K. Johanson et al., The Journal ofBiological Chemistry, Vol. 270, No. 16:9459-9471 (1995).

As mentioned, DR4 polypeptides may be modified by either naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given DR4 polypeptide.Also, a given DR4 polypeptide may contain many types of modifications.DR4 polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic DR4 polypeptides may result fromposttranslational natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

The DR4 can be recovered and purified from chemical synthesis andrecombinant cell cultures by standard methods which include, but are notlimited to, ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxyapatite chromatography and lectin chromatography.Most preferably, high performance liquid chromatography (“HPLC”) isemployed for purification.

DR4 polynucleotides and polypeptides may be used in accordance with thepresent invention for a variety of applications, particularly those thatmake use of the chemical and biological properties of DR4. Among theseare applications in the treatment and prevention of tumors, parasiticinfections, bacterial infections, viral infections, restenosis, andgraft vs. host disease; to induce resistance to parasites, bacteria andviruses; to induce proliferation of T-cells, endothelial cells andcertain hematopoietic cells; to regulate anti-viral responses; and totreat and prevent certain autoimmune diseases after stimulation of DR4by an agonist. Additional applications relate to diagnosis, treatment,and prevention of disorders of cells, tissues and organisms. Theseaspects of the invention are discussed further below.

DR4Proteins and Fragments

The invention further provides for DR4 proteins containing polypeptidesequences encoded by the polynucleotides of the invention.

The DR4 proteins of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers, and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the DR4 proteinsof the invention, their preparation, and compositions (preferably,pharmaceutical compositions) containing them. In specific embodiments,the polypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing only DR4proteins of the invention (including DR4 fragments, variants, and fusionproteins, as described herein). These homomers may contain DR4 proteinshaving identical or different polypeptide sequences. In a specificembodiment, a homomer of the invention is a multimer containing only DR4proteins having an identical polypeptide sequence. In another specificembodiment, a homomer of the invention is a multimer containing DR4proteins having different polypeptide sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing DR4 proteins having identical or different polypeptidesequences) or a homotrimer (e.g., containing DR4 proteins havingidentical or different polypeptide sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous proteins (i.e., proteins containing only polypeptidesequences that do not correspond to a polypeptide sequences encoded bythe DR4 gene) in addition to the DR4 proteins of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when proteins of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when proteins of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the DR4 proteins of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence of the protein (e.g., thepolypeptide sequence recited in SEQ ID NO: 2 or the polypeptide encodedby the deposited cDNA). In one instance, the covalent associations arecross-linking between cysteine residues located within the polypeptidesequences of the proteins which interact in the native (i.e., naturallyoccurring) polypeptide. In another instance, the covalent associationsare the consequence of chemical or recombinant manipulation.Alternatively, such covalent associations may involve one or more aminoacid residues contained in the heterologous polypeptide sequence in aDR4 fusion protein. In one example, covalent associations are betweenthe heterologous sequence contained in a fusion protein of the invention(see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained ina DR4-Fc fusion protein of the invention (as described herein). Inanother specific example, covalent associations of fusion proteins ofthe invention are between heterologous polypeptide sequences fromanother TNF family ligand/receptor member that is capable of formingcovalently associated multimers, such as for example, oseteoprotegerin(see, e.g., International Publication No. WO 98/49305, the contents ofwhich are herein incorporated by reference in its entirety).

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, proteins desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the polypeptidesequence of the proteins desired to be contained in the multimer (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety). Further, proteins of the invention may be routinelymodified by the addition of cysteine or biotin to the C-terminus orN-terminus of the polypeptide sequence of the protein and techniquesknown in the art may be applied to generate multimers containing one ormore of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925,which is herein incorporated by reference in its entirety).Additionally, techniques known in the art may be applied to generateliposomes containing the protein components desired to be contained inthe multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art.

In one embodiment, proteins contained in multimers of the invention areproduced recombinantly using fusion protein technology described hereinor otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain andwhich can be incorporated by membrane reconstitution techniques intoliposomes (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Accordingly,in one embodiment, the invention further provides an isolated DR4polypeptide having the amino acid sequence encoded by the depositedcDNA, or the amino acid sequence shown in SEQ ID NO:2 or a peptide orpolypeptide portion (i.e., fragment) comprising a portion of the abovepolypeptides.

Polypeptide fragments of the present invention include polypeptidescomprising, or alternatively consisting of, an amino acid sequencecontained in SEQ ID NO:2, encoded by the cDNA contained in the depositedplasmid, or encoded by nucleic acids which hybridize (e.g., understringent hybridization conditions) to the nucleotide sequence containedin the deposited plasmid, or shown in SEQ ID NO:1 or the complementarystrand thereto. Protein fragments may be “free-standing,” or comprisedwithin a larger polypeptide of which the fragment forms a part orregion, most preferably as a single continuous region. Representativeexamples of polypeptide fragments of the invention, include, forexample, fragments that comprise, or alternatively consisting of, fromabout amino acid residues: 1 to 23, 24 to 43, 44 to 63, 64 to 83, 84 to103, 104 to 123, 124 to 143, 144 to 163, 164 to 183, 184 to 203, 204 to223, 224 to 238, 239 to 264, 265 to 284, 285 to 304, 305 to 324, 325 to345, 346 to 366, 367 to 387, 388 to 418, 419 to 439, and/or 440 to theend of the coding region of SEQ ID NO:2. Additional representativeexamples of polypeptide fragments of the invention, include, forexample, fragments that comprise, or alternatively consisting of, fromabout amino acid residues: 1-60, 11-70, 21-80, 31-90, 41-100, 51-110,61-120, 71-130, 81-140, 91-150, 101-160, 111-170, 121-180, 131-190,141-200, 151-210, 161-220, 171-230, 181-240, 191-250, 201-260, 211-270,221-280, 231-290, 241-300, 251-310, 261-320, 271-330, 281-340, 291-350,301-360, 311-370, 321-380, 331-390, 341-400, 351-410, 361-420, 371-430,381-440, 391-450, and/or 401-468 of SEQ ID NO:2, as well as isolatedpolynucleotides which encode these polypeptides. In this context “about”includes the particularly recited value, larger or smaller by several(5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.Moreover, polypeptide fragments can be at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acidsin length. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Preferred polypeptide fragments of the present invention include amember selected from the group: a polypeptide comprising, oralternatively consisting of, the DR4 receptor extracellular domain(predicted to constitute amino acid residues from about 24 to about 238in SEQ ID NO:2); a polypeptide comprising, or alternatively consistingof, the DR4 cysteine rich domain (predicted to constitute amino acidresidues from about 131 to about 229 in SEQ ID NO:2); a polypeptidecomprising, or alternatively consisting of, the DR4 receptortransmembrane domain (predicted to constitute amino acid residues fromabout 239 to about 264 in SEQ ID NO:2); a polypeptide comprising, oralternatively consisting of, a fragment of the predicted mature DR4polypeptide, wherein the fragment has a DR4 functional activity (e.g.,antigenic activity or biological activity); a polypeptide comprising, oralternatively consisting of, the DR4 receptor intracellular domain(predicted to constitute amino acid residues from about 265 to about 468in SEQ ID NO:2); a polypeptide comprising, or alternatively consistingof, the DR4 receptor extracellular and intracellular domains with all orpart of the transmembrane domain deleted; a polypeptide comprising, oralternatively consisting of, the DR4 receptor death domain (predicted toconstitute amino acid residues from about 379 to about 422 in SEQ IDNO:2); and a polypeptide comprising, or alternatively consisting of,one, two, three, four or more, epitope bearing portions of the DR4receptor protein. In additional embodiments, the polypeptide fragmentsof the invention comprise, or alternatively consist of, any combinationof 1, 2, 3, 4, 5, 6, 7, or all 8 of the above members. As above, withthe leader sequence, the amino acid residues constituting the DR4receptor extracellular, transmembrane and intracellular domains havebeen predicted by computer analysis. Thus, as one of ordinary skillwould appreciate, the amino acid residues constituting these domains mayvary slightly (e.g., by about 1 to about 15 amino acid residues)depending on the criteria used to define each domain. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

As discussed above, it is believed that one or both of the extracellularcysteine rich motifs of DR4 is important for interactions between DR4and its ligands (e.g., TRAIL). Accordingly, in preferred embodiments,polypeptide fragments of the invention comprise, or alternativelyconsist of, amino acid residues 131 to 183, and/or 184 to 229 of SEQ IDNO:2. In a specific embodiment the polypeptides of the inventioncomprise, or alternatively consist of, both of the extracellularcysteine rich motifs disclosed in SEQ ID NO:2. Polynucleotides encodingthese polypeptide fragments are also encompassed by the invention.

Among the especially preferred polypeptide fragments of the inventionare fragments comprising, or alternatively consisting of, structural orfunctional attributes of DR4. Such fragments include amino acid residuesthat comprise, or alternatively consisting of, one, two, three, four ormore of the following functional domains: alpha-helix and alpha-helixforming regions (“alpha-regions”), beta-sheet and beta-sheet-formingregions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, surface forming regions, and high antigenicindex regions (i.e., containing four or more contiguous amino acidshaving an antigenic index of greater than or equal to 1.5, as identifiedusing the default parameters of the Jameson-Wolf program) of complete(i.e., full-length) DR4. Certain preferred regions are those set out inFIG. 3 and Table I and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequencedepicted in SEQ ID NO:2, such preferred regions include; Garnier-Robsonpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions;Kyte-Doolittle predicted hydrophilic regions; Eisenberg alpha and betaamphipathic regions; Emini surface-forming regions; and Jameson-Wolfhigh antigenic index regions, as predicted using the default parametersof these computer programs. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in the cDNAassigned ATCC Accession No. 97853, encoded by a polynucleotide thathybridizes to the complement of the sequence of SEQ ID NO:1, orcontained in the cDNA assigned ATCC Accession No. 97853 under stringenthybridization conditions or lower stringency hybridization conditions asdefined supra. The present invention further encompasses polynucleotidesequences encoding an epitope of a polypeptide sequence of the invention(such as, for example, the sequence disclosed in SEQ ID NO:1),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments that function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777.

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids contained within the amino acid sequence of a polypeptide ofthe invention. Preferred polypeptides comprising immunogenic orantigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.Antigenic epitopes are useful, for example, to raise antibodies,including monoclonal antibodies, that specifically bind the epitope.Antigenic epitopes can be used as the target molecules in immunoassays.(See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe etal., Science 219:660-666 (1983)). Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). A preferred immunogenic epitope includes thesecreted protein. The polypeptides comprising one or more immunogenicepitopes may be presented for eliciting an antibody response togetherwith a carrier protein, such as an albumin, to an animal system (suchas, for example, rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as, for example, rabbits, rats, and miceare immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 micrograms of peptide or carrier protein andFreund's adjuvant or any other adjuvant known for stimulating an immuneresponse. Several booster injections may be needed, for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody that can be detected, for example, by ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate DR4-specific antibodies include: a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 35 to about 92 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 114 to about160 in SEQ ID NO:2; a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 169 to about 240 in SEQ IDNO:2; a polypeptide comprising, or alternatively consisting of, aminoacid residues from about 267 to about 298 in SEQ ID NO:2; a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 330 to about 364 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 391 to about404 in SEQ ID NO:2; and a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 418 to about 465 in SEQ IDNO:2. In this context “about” includes the particularly recited range,larger or smaller by several (5, 4, 3, 2, or 1) amino acid residues, ateither terminus or at both termini. As indicated above, the inventorshave determined that the above polypeptide fragments are antigenicregions of the DR4 protein. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). IgG Fusion proteins thathave a disulfide-linked dimeric structure due to the IgG portiondisulfide bonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo andBlasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide coding apolypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

As one of skill in the art will appreciate, DR4 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedherein (e.g., corresponding to a portion of the extracellular domainsuch as, for example, amino acid residues 1 to 240 of SEQ ID NO:2) canbe combined with parts of the constant domain of immunoglobulins (IgG),resulting in chimeric polypeptides. These fusion proteins facilitatepurification and show an increased half-life in vivo. This has beenshown, e.g., for chimeric proteins consisting of the first two domainsof the human CD4-polypeptide and various domains of the constant regionsof the heavy or light chains of mammalian immunoglobulins (EPA 394,827;Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericDR4 protein or protein fragment alone (Fountoulakis et al., J Biochem270:3958-3964 (1995)).

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. Houghten, R. A., “General method forthe rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

To improve or alter the characteristics of DR4 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins” including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993)reported modified KGF proteins that had heparin binding activity even if3, 8, or 27 amino-terminal amino acid residues were missing. In thepresent case, since the protein of the invention is a member of thedeath domain containing receptor (DDCR) polypeptide family, deletions ofN-terminal amino acids up to the cysteine residue at position 132 in SEQID NO:2 may retain some biological activity such as the ability toinduce apoptosis. Polypeptides having further N-terminal deletionsincluding the cysteine residue at position 132 (C-132) in SEQ ID NO:2would not be expected to retain such biological activities because thisresidue is conserved among family members, see FIG. 2, and may berequired for forming a disulfide bridge to provide structural stabilitywhich is needed for receptor binding.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification or loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind DR4 ligand (e.g.,TRAIL)) may still be retained. For example, the ability of shortened DR4muteins to induce and/or bind to antibodies which recognize the completeor mature forms of the DR4 polypeptides of the invention (preferablyantibodies that bind specifically to DR4) generally will be retainedwhen less than the majority of the residues of the complete or maturepolypeptide are removed from the N-terminus. Whether a particularpolypeptide lacking N-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that an DR4 mutein with a large number of deleted N-terminalamino acid residues may retain some biological or immunogenicactivities. In fact, peptides composed of as few as six DR4 amino acidresidues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the DR4 aminoacid sequence shown in SEQ ID NO:2, up to the serine residue at positionnumber 463 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues n¹-468of SEQ ID NO:2, where n¹ is an integer from 2 to 463 corresponding tothe position of the amino acid residue in SEQ ID NO:2.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of A-2 to E-468; P-3 to E-468; P4 to E-468; P-5 toE-468; A-6 to E-468; R-7 to E-468; V-8 to E-468; H-9 to E-468; L-10 toE-468; G-11 to E-468; A-12 to E-468; F-13 to E-468; L-14 to E-468; A-15to E-468; V-16 to E-468; T-17 to E-468; P-18 to E-468; N-19 to E-468;P-20 to E-468; G-21 to E-468; S-22 to E-468; A-23 to E-468; A-24 toE-468; S-25 to E-468; G-26 to E-468; T-27 to E-468; E-28 to E-468; A-29to E-468; A-30 to E-468; A-31 to E-468; A-32 to E-468; T-33 to E-468;P-34 to E-468; S-35 to E-468; K-36 to E-468; V-37 to E-468; W-38 toE-468; G-39 to E-468; S40 to E-468; S-41 to E-468; A-42 to E-468; G-43to E-468; R-44 to E-468; 1-45 to E-468; E-46 to E-468; P47 to E-468;R-48 to E-468; G-49 to E-468; G-50 to E-468; G-51 to E-468; R-52 toE-468; G-53 to E-468; A-54 to E-468; L-55 to E-468; P-56 to E-468; T-57to E-468; S-58 to E-468; M-59 to E-468; G-60 to E-468; Q-61 to E-468;H-62 to E-468; G-63 to E-468; P-64 to E-468; S-65 to E-468; A-66 toE-468; R-67 to E-468; A-68 to E-468; R-69 to E-468; A-70 to E-468; G-71to E-468; R-72 to E-468; A-73 to E-468; P-74 to E-468; G-75 to E-468;P-76 to E-468; R-77 to E-468; P-78 to E-468; A-79 to E-468; R-80 toE-468; E-81 to E-468; A-82 to E-468; S-83 to E-468; P-84 to E-468; R-85to E-468; L-86 to E-468; R-87 to E-468; V-88 to E-468; H-89 to E-468;K-90 to E-468; T-91 to E-468; F-92 to E-468; K-93 to E-468; F-94 toE-468; V-95 to E-468; V-96 to E-468; V-97 to E-468; G-98 to E-468; V-99to E-468; L-100 to E-468; L-101 to E-468; Q-102 to E-468; V-103 toE-468; V-104 to E-468; P-105 to E-468; S-106 to E-468; S-107 to E-468;A-108 to E-468; A-109 to E-468; T-110 to E-468; I-111 to E-468; K-112 toE-468; L-113 to E-468; H-114 to E-468; D-115 to E-468; Q-116 to E-468;S-117 to E-468; 1-118 to E-468; G-119 to E-468; T-120 to E-468; Q-121 toE-468; Q-122 to F-468; W-123 to E-468; E-124 to F-468; H-125 to F-468;S-126 to E-468; P-127 to E-468; L-128 to E-468; G-129 to E-468; E-130 toE-468; L-131 to E-468; C-132 to E-468; P-133 to E-468; P-134 to E-468;G-135 to E-468; S-136 to E-468; H-137 to E-468; R-138 to E-468; S-139 toE-468; E-140 to E-468; R-141 to E-468; P-142 to E-468; G-143 to E-468;A-144 to E-468; C-145 to E-468; N-146 to E-468; R-147 to E-468; C-148 toE-468; T-149 to E-468; E-150 to E-468; G-151 to E-468; V-152 to E-468;G-153 to E-468; Y-154 to E-468; T-155 to E-468; N-156 to E-468; A-157 toE-468; S-158 to E-468; N-159 to E-468; N-160 to E-468; L-161 to E-468;F-162 to E-468; A-163 to E-468; C-164 to E-468; L-165 to E-468; P-166 toE-468; C-167 to E-468; T-168 to E-468; A-169 to E-468; C-170 to E-468;K-171 to E-468S; S-172 to E-468; D-173 to E-468; E-174 to E-468; E-175to E-468; E-176 to E-468; R-177 to E-468; S-178 to E-468; P-179 toE-468; C-180 to E-468; T-181 to E-468; T-182 to E-468; T-183 to E-468;R-184 to E-468; N-185 to E-468; T-186 to E-468; A-187 to E-468; C-188 toE-468; Q-189 to E-468; C-190 to E-468; K-191 to E-468; P-192 to E-468;G-193 to E-468; T-194 to E-468; F-195 to E-468; R-196 to E-468; N-197 toE-468; D-198 to E-468; N-199 to E-468; S-200 to E-468; A-201 to E-468;E-202 to E-468; M-203 to E-468; C-204 to E-468; R-205 to E-468; K-206 toE-468; C-207 to E-468; S-208 to E-468; T-209 to E-468; G-210 to E-468;C-211 to E-468; P-212 to E-468; R-213 to E-468; G-214 to E-468; M-215 toE-468; V-216 to E-468; K-217 to E-468; V-218 to E-468; K-219 to E-468;D-220 to E-468; C-221 to E-468; T-222 to E-468; P-223 to E-468; W-224 toE-468; S-225 to E-468; D-226 to E-468; 1-227 to E-468; E-228 to E-468;C-229 to E-468; V-230 to E-468; H-231 to E-468; K-232 to E-468; E-233 toE-468; S-234 to E-468; G-235 to E-468; N-236 to E-468; G-237 to E-468;H-238 to E-468; N-239 to E-468; 1-240 to E-468; W-241 to E-468; V-242 toE-468; 1-243 to E-468; L-244 to E-468; V-245 to E-468; V-246 to E-468;T-247 to E-468; L-248 to E-468; V-249 to E-468; V-250 to E-468; P-251 toE-468; L-252 to E-468; L-253 to E-468; L-254 to E-468; V-255 to E-468;A-256 to E-468; V-257 to E-468; L-258 to E-468; 1-259 to E-468; V-260 toE-468; C-261 to E-468; C-262 to E-468; C-263 to E-468; 1-264 to E-468;G-265 to E-468; S-266 to E-468; G-267 to E-468; C-268 to E-468; G-269 toE-468; G-270 to E-468; D-271 to E-468; P-272 to E-468; K-273 to E-468;C-274 to E-468; M-275 to E-468; D-276 to E-468; R-277 to E-468; V-278 toE-468; C-279 to E-468; F-280 to E-468; W-281 to E-468; R-282 to E-468;L-283 to E-468; G-284 to E-468; L-285 to E-468; L-286 to E-468; R-287 toE-468; G-288 to E-468; P-289 to E-468; G-290 to E-468; A-291 to E-468;E-292 to E-468; D-293 to E-468; N-294 to E-468; A-295 to E-468; H-296 toE-468; N-297 to E-468; E-298 to E-468; 1-299 to E-468; L-300 to E-468;S-301 to E-468; N-302 to E-468; A-303 to E-468; D-304 to E-468; S-305 toE-468; L-306 to E-468; S-307 to E-468; T-308 to E-468; F-309 to E-468;V-310 to E-468; S-311 to E-468; E-312 to E-468; Q-313 to E-468; Q-314 toE-468; M-315 to E-468; E-316 to E-468; S-317 to E-468; Q-318 to E-468;E-319 to E-468; P-320 to E-468; A-321 to E-468; D-322 to E-468; L-323 toE-468; T-324 to E-468; G-325 to E-468; V-326 to E-468; T-327 to E-468;V-328 to E-468; E-329 to E-468; S-330 to E-468; P-331 to E-468; G-332 toE-468; E-333 to E-468; A-334 to E-468; Q-335 to E-468; C-336 to E-468;L-337 to E-468; L-338 to E-468; G-339 to E-468; P-340 to E-468; A-341 toE-468; E-342 to E-468; A-343 to E-468; E-344 to E-468; G-345 to E-468;S-346 to E-468; Q-347 to E-468; R-348 to E-468; R-349 to E-468; R-350 toE-468; L-351 to E-468; L-352 to E-468; V-353 to E-468; P-354 to E-468;A-355 to E-468; N-356 to E-468; G-357 to E-468; A-358 to E-468; D-359 toE-468; P-360 to E-468; T-361 to E-468; E-362 to E-468; T-363 to E-468;L-364 to E-468; M-365 to E-468; L-366 to E-468; F-367 to E-468; F-368 toE-468; D-369 to E-468; K-370 to E-468; F-371 to E-468; A-372 to E-468;N-373 to E-468; 1-374 to E-468; V-375 to E-468; P-376 to E-468; F-377 toE-468; D-378 to E-468; S-379 to E-468; W-380 to E-468; D-381 to E-468;Q-382 to E-468; L-383 to E-468; M-384 to E-468; R-385 to E-468; Q-386 toE-468; L-387 to E-468; D-388 to E-468; L-389 to E-468; T-390 to E-468;K-391 to E-468; N-392 to E-468; E-393 to E-468; 1-394 to E-468; D-395 toE-468; V-396 to E-468; V-397 to E-468; R-398 to E-468; A-399 to E-468;G-400 to E-468; T-401 to E-468; A-402 to E-468; G-403 to E-468; P404 toE-468; G-405 to E-468; D-406 to E-468; A-407 to E-468; L-408 to E-468;Y-409 to E-468; A-410 to E-468; M-411 to E-468; L-412 to E-468; M-413 toE-468; K-414 to E-468; W-415 to E-468; V-416 to E-468; N-417 to E-468;K-418 to E-468; T-419 to E-468; G-420 to E-468; R-421 to E-468; N-422 toE-468; A-423 to E-468; S424 to E-468; 1-425 to E-468; H-426 to E-468;T-427 to E-468; L-428 to E-468; L-429 to E-468; D-430 to E-468; A-431 toE-468; L-432 to E-468; E-433 to E-468; R-434 to E-468; M-435 to E-468;E-436 to E-468; E-437 to E-468; R-438 to E-468; H-439 to E-468; A-440 toE-468; K-441 to E-468; E-442 to E-468; K-443 to E-468; 1-444 to E-468;Q-445 to E-468; D-446 to E-468; L-447 to E-468; L-448 to E-468; V-449 toE-468; D-450 to E-468; S451 to E-468; G-452 to E-468; K-453 to E-468;F-454 to E-468; 1-455 to E-468; Y-456 to E-468; L-457 to E-468; E-458 toE-468; D-459 to E-468; G-460 to E-468; T-461 to E-468; G-462 to E-468;and S-463 to E-468 of the DR4 sequence shown in SEQ ID NO:2.

The present invention is also directed to nucleic acid moleculescomprising, or alternatively consisting of, a polynucleotide sequence atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequences encoding the polypeptides described above. Theinvention is further directed to nucleic acid molecules comprising, oralternatively consisting of, polynucleotide sequences which encodepolypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,or 99% identical to the polypeptides described above. The presentinvention also encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

In another embodiment, N-terminal deletions of the DR4 polypeptide canbe described by the general formula n² to 238 where n² is a number from2 to 238 corresponding to the amino acid sequence identified in SEQ IDNO:2. In specific embodiments, N-terminal deletions of the DR4 receptorsof the invention provides polynucleotides encoding polypeptidescomprising, or alternatively consisting of, the amino acid sequence ofresidues: A-2 to H-238; P-3 to H-238; P4 to H-238; P-5 to H-238; A-6 toH-238; R-7 to H-238; V-8 to H-238; H-9 to H-238; L-10 to H-238; G-11 toH-238; A-12 to H-238; F-13 to H-238; L-14 to H-238; A-15 to H-238; V-16to H-238; T-17 to H-238; P-18 to H-238; N-19 to H-238; P-20 to H-238;G-21 to H-238; S-22 to H-238; A-23 to H-238; A-24 to H-238; S-25 toH-238; G-26 to H-238; T-27 to H-238; E-28 to H-238; A-29 to H-238; A-30to H-238; A-31 to H-238; A-32 to H-238; T-33 to H-238; P-34 to H-238;S-35 to H-238; K-36 to H-238; V-37 to H-238; W-38 to H-238; G-39 toH-238; S-40 to H-238; S41 to H-238; A-42 to H-238; G-43 to H-238; R-44to H-238; 1-45 to H-238; E-46 to H-238; P-47 to H-238; R-48 to H-238;G-49 to H-238; G-50 to H-238; G-51 to H-238; R-52 to H-238; G-53 toH-238; A-54 to H-238; L-55 to H-238; P-56 to H-238; T-57 to H-238; S-58to H-238; M-59 to H-238; G-60 to H-238; Q-61 to H-238; H-62 to H-238;G-63 to H-238; P-64 to H-238; S-65 to H-238; A-66 to H-238; R-67 toH-238; A-68 to H-238; R-69 to H-238; A-70 to H-238; G-71 to H-238; R-72to H-238; A-73 to H-238; P-74 to H-238; G-75 to H-238; P-76 to H-238;R-77 to H-238; P-78 to H-238; A-79 to H-238; R-80 to H-238; E-81 toH-238; A-82 to H-238; S-83 to H-238; P-84 to H-238; R-85 to H-238; L-86to H-238; R-87 to H-238; V-88 to H-238; H-89 to H-238; K-90 to H-238;T-91 to H-238; F-92 to H-238; K-93 to H-238; F-94 to H-238; V-95 toH-238; V-96 to H-238; V-97 to H-238; G-98 to H-238; V-99 to H-238; L-100to H-238; L-101 to H-238; Q-102 to H-238; V-103 to H-238; V-104 toH-238; P-105 to H-238; S-106 to H-238; S-107 to H-238; A-108 to H-238;A-109 to H-238; T-110 to H-238; 1-111 to H-238; K-112 to H-238; L-113 toH-238; H-114 to H-238; D-115 to H-238; Q-116 to H-238; S-117 to H-238;1-118 to H-238; G-119 to H-238; T-120 to H-238; Q-121 to H-238; Q-122 toH-238; W-123 to H-238; E-124 to H-238; H-125 to H-238; S-126 to H-238;P-127 to H-238; L-128 to H-238; G-129 to H-238; E-130 to H-238; L-131 toH-238; C-132 to H-238; P-133 to H-238; P-134 to H-238; G-135 to H-238;S-136 to H-238; H-137 to H-238; R-138 to H-238; S-139 to H-238; E-140 toH-238; R-141 to H-238; P-142 to H-238; G-143 to H-238; A-144 to H-238;C-145 to H-238; N-146 to H-238; R-147 to H-238; C-148 to H-238; T-149 toH-238; E-150 to H-238; G-151 to H-238; V-152 to H-238; G-153 to H-238;Y-154 to H-238; T-155 to H-238; N-156 to H-238; A-157 to H-238; S-158 toH-238; N-159 to H-238; N-160 to H-238; L-161 to H-238; F-162 to H-238;A-163 to H-238; C-164 to H-238; L-165 to H-238; P-166 to H-238; C-167 toH-238; T-168 to H-238; A-169 to H-238; C-170 to H-238; K-171 to H-238;S-172 to H-238; D-173 to H-238; E-174 to H-238; E-175 to H-238; E-176 toH-238; R-177 to H-238; S-178 to H-238; P-179 to H-238; C-180 to H-238;T-181 to H-238; T-182 to H-238; T-183 to H-238; R-184 to H-238; N-185 toH-238; T-186 to H-238; A-187 to H-238; C-188 to H-238; Q-189 to H-238;C-190 to H-238; K-191 to H-238; P-192 to H-238; G-193 to H-238; T-194 toH-238; F-195 to H-238; R-196 to H-238; N-197 to H-238; D-198 to H-238;N-199 to H-238; S-200 to H-238; A-201 to H-238; E-202 to H-238; M-203 toH-238; C-204 to H-238; R-205 to H-238; K-206 to H-238; C-207 to H-238;S-208 to H-238; T-209 to H-238; G-210 to H-238; C-211 to H-238; P-212 toH-238; R-213 to H-238; G-214 to H-238; M-215 to H-238; V-216 to H-238;K-217 to H-238; V-218 to H-238; K-219 to H-238; D-220 to H-238; C-221 toH-238; T-222 to H-238; P-223 to H-238; W-224 to H-238; S-225 to H-238;D-226 to H-238; 1-227 to H-238; E-228 to H-238; C-229 to H-238; V-230 toH-238; H-231 to H-238; K-232 to H-238; and E-233 to H-238 of the DR4extracellular domain sequence shown in SEQ ID NO:2.

The present invention is also directed to nucleic acid moleculescomprising, or alternatively consisting of, a polynucleotide sequence atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequences encoding the polypeptides described above. Theinvention is further directed to nucleic acid molecules comprising, oralternatively consisting of, polynucleotide sequences which encodepolypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,or 99% identical to the polypeptides described above. The presentinvention also encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

Similarly, many examples of functional C-terminal deletion muteins areknown. For instance, interferon gamma shows up to ten times higheractivities by deleting 8-10 amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. Biotechnology 7:199-216(1988). In the present case, since the protein of the invention is amember of the DDCR polypeptide family, deletions of C-terminal aminoacids up to the cysteine at position 221 (C-221) of SEQ ID NO:2 mayretain some biological activity such as receptor binding. Polypeptideshaving further C-terminal deletions including C-221 of SEQ ID NO:2 wouldnot be expected to retain such biological activities because thisresidue is conserved among DDCR family members and is required forforming a disulfide bridge to provide structural stability which isneeded for receptor-ligand binding.

As mentioned above, even if deletion of one or more amino acids from theC-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other functional activities (e.g.,biological activities, ability to multimerize, ability to bind DR4ligand (e.g., TRAIL)) may still be retained. For example the ability ofthe shortened DR4 mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a DR4 mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as six DR4amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the DR4 polypeptide shown in SEQ ID NO:2, up to the alanineresidue at position number 30, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising, or alternatively consisting of, the amino acid sequence ofresidues 24-m¹ of SEQ ID NO:2, where m¹ is an integer from 30 to 467corresponding to the position of the amino acid residue in SEQ ID NO:2.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues A-24 to L-467; A-24 to S-466; A-24 to V-465; A-24to A-464; A-24 to S-463; A-24 to G-462; A-24 to T-461; A-24 to G-460;A-24 to D-459; A-24 to E-458; A-24 to L-457; A-24 to Y-456; A-24 toI-455; A-24 to F-454; A-24 to K-453; A-24 to G-452; A-24 to S-451; A-24to D-450; A-24 to V-449; A-24 to L-448; A-24 to L-447; A-24 to D-446;A-24 to Q-445; A-24 to I-444; A-24 to K-443; A-24 to E-442; A-24 toK-441; A-24 to A-440; A-24 to H-439; A-24 to R-438; A-24 to E-437; A-24to E-436; A-24 to M-435; A-24 to R-434; A-24 to E-433; A-24 to L-432;A-24 to A-431; A-24 to D-430; A-24 to L-429; A-24 to L-428; A-24 toT-427; A-24 to H-426; A-24 to I-425; A-24 to S-424; A-24 to A-423; A-24to N-422; A-24 to R-421; A-24 to G-420; A-24 to T-419; A-24 to K-418;A-24 to N-417; A-24 to V-416; A-24 to W-415; A-24 to K-414; A-24 toM-413; A-24 to L-412; A-24 to M-411; A-24 to A-410; A-24 to Y-409; A-24to L-408; A-24 to A-407; A-24 to D-406; A-24 to G-405; A-24 to P-404;A-24 to G-403; A-24 to A-402; A-24 to T-401; A-24 to G-400; A-24 toA-399; A-24 to R-398; A-24 to V-397; A-24 to V-396; A-24 to D-395; A-24to I-394; A-24 to E-393; A-24 to N-392; A-24 to K-391; A-24 to T-390;A-24 to L-389; A-24 to D-388; A-24 to L-387; A-24 to Q-386; A-24 toR-385; A-24 to M-384; A-24 to L-383; A-24 to Q-382; A-24 to D-381; A-24to W-380; A-24 to S-379; A-24 to D-378; A-24 to F-377; A-24 to P-376;A-24 to V-375; A-24 to I-374; A-24 to N-373; A-24 to A-372; A-24 toF-371; A-24 to K-370; A-24 to D-369; A-24 to F-368; A-24 to F-367; A-24to L-366; A-24 to M-365; A-24 to L-364; A-24 to T-363; A-24 to E-362;A-24 to T-361; A-24 to P-360; A-24 to D-359; A-24 to A-358; A-24 toG-357; A-24 to N-356; A-24 to A-355; A-24 to P-354; A-24 to V-353; A-24to L-352; A-24 to L-351; A-24 to R-350; A-24 to R-349; A-24 to R-348;A-24 to Q-347; A-24 to S-346; A-24 to G-345; A-24 to E-344; A-24 toA-343; A-24 to E-342; A-24 to A-341; A-24 to P-340; A-24 to G-339; A-24to L-338; A-24 to L-337; A-24 to C-336; A-24 to Q-335; A-24 to A-334;A-24 to E-333; A-24 to G-332; A-24 to P-331; A-24 to S-330; A-24 toQ-329; A-24 to V-328; A-24 to T-327; A-24 to V-326; A-24 to G-325; A-24to T-324; A-24 to L-323; A-24 to D-322; A-24 to A-321; A-24 to P-320;A-24 to E-319; A-24 to Q-318; A-24 to S-317; A-24 to E-316; A-24 toM-315; A-24 to Q-314; A-24 to Q-313; A-24 to E-312; A-24 to S-311; A-24to V-310; A-24 to F-309; A-24 to T-308; A-24 to S-307; A-24 to L-306;A-24 to S-305; A-24 to D-304; A-24 to A-303; A-24 to N-302; A-24 toS-301; A-24 to L-300; A-24 to I-299; A-24 to E-298; A-24 to N-297; A-24to H-296; A-24 to A-295; A-24 to N-294; A-24 to D-293; A-24 to E-292;A-24 to A-291; A-24 to G-290; A-24 to P-289; A-24 to G-288; A-24 toR-287; A-24 to L-286; A-24 to L-285; A-24 to G-284; A-24 to L-283; A-24to R-282; A-24 to W-281; A-24 to F-280; A-24 to C-279; A-24 to V-278;A-24 to R-277; A-24 to D-276; A-24 to M-275; A-24 to C-274; A-24 toK-273; A-24 to P-272; A-24 to D-271; A-24 to G-270; A-24 to G-269; A-24to C-268; A-24 to G-267; A-24 to S-266; A-24 to G-265; A-24 to I-264;A-24 to C-263; A-24 to C-262; A-24 to C-261; A-24 to V-260; A-24 toI-259; A-24 to L-258; A-24 to V-257; A-24 to A-256; A-24 to V-255; A-24to L-254; A-24 to L-253; A-24 to L-252; A-24 to P-251; A-24 to V-250;A-24 to V-249; A-24 to L-248; A-24 to T-247; A-24 to V-246; A-24 toV-245; A-24 to L-244; A-24 to I-243; A-24 to V-242; A-24 to W-241; A-24to I-240; A-24 to N-239; A-24 to H-238; A-24 to G-237; A-24 to N-236;A-24 to G-235; A-24 to S-234; A-24 to E-233; A-24 to K-232; A-24 toH-231; A-24 to V-230; A-24 to C-229; A-24 to E-228; A-24 to I-227; A-24to D-226; A-24 to S-225; A-24 to W-224; A-24 to P-223; A-24 to T-222;A-24 to C-221; A-24 to D-220; A-24 to K-219; A-24 to V-218; A-24 toK-217; A-24 to V-216; A-24 to M-215; A-24 to G-214; A-24 to R-213; A-24to P-212; A-24 to C-211; A-24 to G-210; A-24 to T-209; A-24 to S-208;A-24 to C-207; A-24 to K-206; A-24 to R-205; A-24 to C-204; A-24 toM-203; A-24 to E-202; A-24 to A-201; A-24 to S-200; A-24 to N-199; A-24to D-198; A-24 to N-197; A-24 to R-196; A-24 to F-195; A-24 to T-194;A-24 to G-193; A-24 to P-192; A-24 to K-191; A-24 to C-190; A-24 toQ-189; A-24 to C-188; A-24 to A-187; A-24 to T-186; A-24 to N-185; A-24to R-184; A-24 to T-183; A-24 to T-182; A-24 to T-181; A-24 to C-180;A-24 to P-179; A-24 to S-178; A-24 to R-177; A-24 to E-176; A-24 toE-175; A-24 to E-174; A-24 to D-173; A-24 to S-172; A-24 to K-171; A-24to C-170; A-24 to A-169; A-24 to T-168; A-24 to C-167; A-24 to P-166;A-24 to L-165; A-24 to C-164; A-24 to A-163; A-24 to F-162; A-24 toL-161; A-24 to N-160; A-24 to N-159; A-24 to S-158; A-24 to A-157; A-24to N-156; A-24 to T-155; A-24 to Y-154; A-24 to G-153; A-24 to V-152;A-24 to G-151; A-24 to E-150; A-24 to T-149; A-24 to C-148; A-24 toR-147; A-24 to N-146; A-24 to C-145; A-24 to A-144; A-24 to G-143; A-24to P-142; A-24 to R-141; A-24 to E-140; A-24 to S-139; A-24 to R-138;A-24 to H-137; A-24 to S-136; A-24 to G-135; A-24 to P-134; A-24 toP-133; A-24 to C-132; A-24 to L-131; A-24 to E-130; A-24 to G-129; A-24to L-128; A-24 to P-127; A-24 to S-126; A-24 to H-125; A-24 to E-124;A-24 to W-123; A-24 to Q-122; A-24 to Q-121; A-24 to T-120; A-24 toG-119; A-24 to I-118; A-24 to S-117; A-24 to Q-116; A-24 to D-115; A-24to H-114; A-24 to L-113; A-24 to K-112; A-24 to I-111; A-24 to T-110;A-24 to A-109; A-24 to A-108; A-24 to S-107; A-24 to S-106; A-24 toP-105; A-24 to V-104; A-24 to V-103; A-24 to Q-102; A-24 to L-101; A-24to L-100; A-24 to V-99; A-24 to G-98; A-24 to V-97; A-24 to V-96; A-24to V-95; A-24 to F-94; A-24 to K-93; A-24 to F-92; A-24 to T-91; A-24 toK-90; A-24 to H-89; A-24 to V-88; A-24 to R-87; A-24 to L-86; A-24 toR-85; A-24 to P-84; A-24 to S-83; A-24 to A-82; A-24 to E-81; A-24 toR-80; A-24 to A-79; A-24 to P-78; A-24 to R-77; A-24 to P-76; A-24 toG-75; A-24 to P-74; A-24 to A-73; A-24 to R-72; A-24 to G-71; A-24 toA-70; A-24 to R-69; A-24 to A-68; A-24 to R-67; A-24 to A-66; A-24 toS-65; A-24 to P-64; A-24 to G-63; A-24 to H-62; A-24 to Q-61; A-24 toG-60; A-24 to M-59; A-24 to S-58; A-24 to T-57; A-24 to P-56; A-24 toL-55; A-24 to A-54; A-24 to G-53; A-24 to R-52; A-24 to G-51; A-24 toG-50; A-24 to G-49; A-24 to R-48; A-24 to P-47; A-24 to E-46; A-24 to145; A-24 to R-44; A-24 to G-43; A-24 to A-42; A-24 to S-41; A-24 toS-40; A-24 to G-39; A-24 to W-38; A-24 to V-37; A-24 to K-36; A-24 toS-35; A-24 to P-34; A-24 to T-33; A-24 to A-32; A-24 to A-31; and A-24to A-30 of the DR4 sequence shown in SEQ ID NO:2.

The present invention is also directed to nucleic acid moleculescomprising, or alternatively consisting of, a polynucleotide sequence atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequences encoding the polypeptides described above. Theinvention is further directed to nucleic acid molecules comprising, oralternatively consisting of, polynucleotide sequences which encodepolypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,or 99% identical to the polypeptides described above. The presentinvention also encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

In another embodiment, C-terminal deletions of the DR4 polypeptide canbe described by the general formula 24-m² where m² is a number from 30to 238 corresponding to the amino acid sequence identified in SEQ IDNO:2. In specific embodiments, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues: A-24 to G-237; A-24 to N-236; A-24 toG-235; A-24 to S-234; A-24 to E-233; A-24 to K-232; A-24 to H-231; A-24to V-230; A-24 to C-229; A-24 to E-228; A-24 to I-227; A-24 to D-226;A-24 to S-225; A-24 to W-224; A-24 to P-223; A-24 to T-222; A-24 toC-221; A-24 to D-220; A-24 to K-219; A-24 to V-218; A-24 to K-217; A-24to V-216; A-24 to M-215; A-24 to G-214; A-24 to R-213; A-24 to P-212;A-24 to C-211; A-24 to G-210; A-24 to T-209; A-24 to S-208; A-24 toC-207; A-24 to K-206; A-24 to R-205; A-24 to C-204; A-24 to M-203; A-24to E-202; A-24 to A-201; A-24 to S-200; A-24 to N-199; A-24 to D-198;A-24 to N-197; A-24 to R-196; A-24 to F-195; A-24 to T-194; A-24 toG-193; A-24 to P-192; A-24 to K-191; A-24 to C-190; A-24 to Q-189; A-24to C-188; A-24 to A-187; A-24 to T-186; A-24 to N-185; A-24 to R-184;A-24 to T-183; A-24 to T-182; A-24 to T-181; A-24 to C-180; A-24 toP-179; A-24 to S-178; A-24 to R-177; A-24 to E-176; A-24 to E-175; A-24to E-174; A-24 to D-173; A-24 to S-172; A-24 to K-171; A-24 to C-170;A-24 to A-169; A-24 to T-168; A-24 to C-167; A-24 to P-166; A-24 toL-165; A-24 to C-164; A-24 to A-163; A-24 to F-162; A-24 to L-161; A-24to N-160; A-24 to N-159; A-24 to S-158; A-24 to A-157; A-24 to N-156;A-24 to T-155; A-24 to Y-154; A-24 to G-153; A-24 to V-152; A-24 toG-151; A-24 to E-150; A-24 to T-149; A-24 to C-148; A-24 to R-147; A-24to N-146; A-24 to C-145; A-24 to A-144; A-24 to G-143; A-24 to P-142;A-24 to R-141; A-24 to E-140; A-24 to S-139; A-24 to R-138; A-24 toH-137; A-24 to S-136; A-24 to G-135; A-24 to P-134; A-24 to P-133; A-24to C-132; A-24 to L-131; A-24 to E-130; A-24 to G-129; A-24 to L-128;A-24 to P-127; A-24 to S-126; A-24 to H-125; A-24 to E-124; A-24 toW-123; A-24 to Q-122; A-24 to Q-121; A-24 to T-120; A-24 to G-119; A-24to I-118; A-24 to S-117; A-24 to Q-116; A-24 to D-115; A-24 to H-114;A-24 to L-113; A-24 to K-112; A-24 to I-111; A-24 to T-110; A-24 toA-109; A-24 to A-108; A-24 to S-107; A-24 to S-106; A-24 to P-105; A-24to V-104; A-24 to V-103; A-24 to Q-102; A-24 to L-101; A-24 to L-100;A-24 to V-99; A-24 to G-98; A-24 to V-97; A-24 to V-96; A-24 to V-95;A-24 to F-94; A-24 to K-93; A-24 to F-92; A-24 to T-91; A-24 to K-90;A-24 to H-89; A-24 to V-88; A-24 to R-87; A-24 to L-86; A-24 to R-85;A-24 to P-84; A-24 to S-83; A-24 to A-82; A-24 to E-81; A-24 to R-80;A-24 to A-79; A-24 to P-78; A-24 to R-77; A-24 to P-76; A-24 to G-75;A-24 to P-74; A-24 to A-73; A-24 to R-72; A-24 to G-71; A-24 to A-70;A-24 to R-69; A-24 to A-68; A-24 to R-67; A-24 to A-66; A-24 to S-65;A-24 to P-64; A-24 to G-63; A-24 to H-62; A-24 to Q-61; A-24 to G-60;A-24 to M-59; A-24 to S-58; A-24 to T-57; A-24 to P-56; A-24 to L-55;A-24 to A-54; A-24 to G-53; A-24 to R-52; A-24 to G-51; A-24 to G-50;A-24 to G-49; A-24 to R-48; A-24 to P-47; A-24 to E-46; A-24 to 145;A-24 to R-44; A-24 to G-43; A-24 to A-42; A-24 to S-41; A-24 to S40;A-24 to G-39; A-24 to W-38; A-24 to V-37; A-24 to K-36; A-24 to S-35;A-24 to P-34; A-24 to T-33; A-24 to A-32; A-24 to A-31; and A-24 to A-30of the DR4 extracellular domain sequence shown in SEQ ID NO:2.

The present invention is also directed to nucleic acid moleculescomprising, or alternatively consisting of, a polynucleotide sequence atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequences encoding the polypeptides described above. Theinvention is further directed to nucleic acid molecules comprising, oralternatively consisting of, polynucleotide sequences which encodepolypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,or 99% identical to the polypeptides described above. The presentinvention also encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

The present invention further provides polypeptides having one or moreresidues from the carboxy terminus of the amino acid sequence of the DR4shown in SEQ ID NO:2, up to C-221 of SEQ ID NO:2, and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides having the amino acid sequence of residues 1-m² ofthe amino acid sequence in SEQ ID NO:2, where m² is any integer in therange of 221-468 and residue C-221 is the position of the first residuefrom the C-terminus of the complete DR4 polypeptide (shown in SEQ IDNO:2) believed to be required for receptor binding activity of the DR4protein. Polynucleotides encoding these polypeptides also are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of an DR4polypeptide, which may be described generally as having residues n¹-m¹and/or n²-m² of SEQ ID NO:2, where n¹, n², m¹, and m² are integers asdescribed above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete DR4 amino acid sequence encodedby the cDNA contained in ATCC Deposit No. 97853, where this portionexcludes from 1 to about 108 amino acids from the amino terminus of thecomplete amino acid sequence encoded by the cDNA contained in ATCCDeposit No. 97853, or from 1 to about 247 amino acids from the carboxyterminus, or any combination of the above amino terminal and carboxyterminal deletions, of the complete amino acid sequence encoded by thecDNA contained in ATCC Deposit No. 97853. Polynucleotides encoding allof the above deletion mutant polypeptide forms also are provided.

Preferred amongst the N- and C-terminal deletion mutants are thosecomprising only a portion of the extracellular domain; i.e., withinresidues 24-238, since any portion therein is expected to be soluble.

It will be recognized in the art that some amino acid sequence of DR4can be varied without significant effect of the structure or function ofthe protein. If such differences in sequence are contemplated, it shouldbe remembered that there will be critical areas on the protein whichdetermine activity. Such areas will usually comprise residues which makeup the ligand binding site or the death domain, or which form tertiarystructures which affect these domains.

Thus, the invention further includes variations of the DR4 protein whichshow substantial DR4 protein activity or which include regions of DR4such as the protein fragments discussed below. Such mutants includedeletions, insertions, inversions, repeats, and type substitutions. Asindicated above, guidance concerning which amino acid changes are likelyto be phenotypically silent can be found in Bowie, J. U. et al., Science247:1306-1310 (1990).

Thus, the fragment, derivative, or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which atleast one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue(s), and more preferably at least one but less thanten conserved amino acid residues) and such substituted amino acidresidue may or may not be one encoded by the genetic code, or (ii) onein which one or more of the amino acid residues includes a substituentgroup, or (iii) one in which the mature polypeptide is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the mature polypeptide, such as anIgG Fc fusion region peptide or leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein. Polynucleotides encoding these fragments, derivativesor analogs are also encompassed by the invention.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the DR4 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-alpha to only one of the two known types of TNF receptors. Thus, theDR4 receptor of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table II).

TABLE II Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

In specific embodiments, the number of substitutions, additions ordeletions in the amino acid sequence of SEQ ID NO:2 and/or any of thepolypeptide fragments described herein (e.g., the extracellular domainor intracellular domain) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10,9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10,1-5, 1-3 or 1-2.

Amino acids in the DR4 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

Additionally, protein engineering may be employed to improve or alterthe characteristics of DR4 polypeptides. Recombinant DNA technologyknown to those skilled in the art can be used to create novel mutantproteins or muteins including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

Thus, the invention also encompasses DR4 derivatives and analogs thathave one or more amino acid residues deleted, added, or substituted togenerate DR4 polypeptides that are better suited for expression, scaleup, etc., in the host cells chosen. For example, cysteine residues canbe deleted or substituted with another amino acid residue in order toeliminate disulfide bridges; N-linked glycosylation sites can be alteredor eliminated to achieve, for example, expression of a homogeneousproduct that is more easily recovered and purified from yeast hostswhich are known to hyperglycosylate N-linked sites. To this end, avariety of amino acid substitutions at one or both of the first or thirdamino acid positions on any one or more of the glycosylationrecognitions sequences in the DR4 polypeptides of the invention, and/oran amino acid deletion at the second position of any one or more suchrecognition sequences will prevent glycosylation of the DR4 at themodified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J.5(6):1193-1197).

The polypeptides of the present invention also include a polypeptidecomprising, or alternatively consisting of, the polypeptide encoded bythe deposited cDNA (the deposit having ATCC Accession No. 97853)including the leader; a polypeptide comprising, or alternativelyconsisting of, the mature polypeptide encoded by the deposited the cDNAminus the leader (i.e., the mature protein); a polypeptide comprising,or alternatively consisting of, the polypeptide of SEQ ID NO:2 includingthe leader; a polypeptide comprising, or alternatively consisting of,the polypeptide of SEQ ID NO:2 minus the amino terminal methionine; apolypeptide comprising, or alternatively consisting of, the polypeptideof SEQ ID NO:2 minus the leader; a polypeptide comprising, oralternatively consisting of, the DR4 extracellular domain; a polypeptidecomprising, or alternatively consisting of, the DR4 cysteine richdomain; a polypeptide comprising, or alternatively consisting of, theDR4 transmembrane domain; a polypeptide comprising, or alternativelyconsisting of, the DR4 intracellular domain; a polypeptide comprising,or alternatively consisting of, the DR4 death domain; a polypeptidecomprising, or alternatively consisting of, soluble polypeptidescomprising all or part of the extracellular and intracellular domainsbut lacking the transmembrane domain; as well as polypeptides which areat least 80% identical, more preferably at least 90% or 95% identical,still more preferably at least 96%, 97%, 98% or 99% identical to thepolypeptides described above (e.g., the polypeptide encoded by thedeposited cDNA, the polypeptide of SEQ ID NO:2, and portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a DR4 polypeptideis intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the DR4 polypeptide. In other words, toobtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence shown in SEQ ID NO:2 or to the aminoacid sequence encoded by the deposited cDNA can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the DR4 polypeptide sequence set forth herein as n¹-m¹,and/or n²-m². In preferred embodiments, the application is directed toproteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% identical to polypeptides having the amino acid sequenceof the specific DR4 N- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In certain preferred embodiments, DR4 proteins of the invention comprisefusion proteins as described above wherein the DR4 polypeptides arethose described as n¹-m¹, and/or n²-m² herein. In preferred embodiments,the application is directed to nucleic acid molecules at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences encoding polypeptides having the amino acid sequence of thespecific N- and C-terminal deletions recited herein. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

The present inventors have discovered that the DR4 polypeptide is a 468residue protein exhibiting three main structural domains. First, theligand binding domain (extracellular domain) was identified withinresidues from about 24 to about 238 in SEQ ID NO:2. Second, thetransmembrane domain was identified within residues from about 239 toabout 264 in SEQ ID NO:2. Third, the intracellular domain was identifiedwithin residues from about 265 to about 468 in SEQ ID NO:2. Importantly,the intracellular domain includes a death domain at residues from about379 to about 422 in SEQ ID NO:2. Further preferred fragments of thepolypeptide shown in SEQ ID NO:2 include the mature protein fromresidues about 24 to about 468 and soluble polypeptides comprising allor part of the extracellular and intracellular domains but lacking thetransmembrane domain. In this context “about” includes the particularlyrecited range, larger or smaller by several (5, 4, 3, 2, or 1) aminoacid residues, at either terminus or at both termini.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide described herein.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. The number of immunogenicepitopes of a protein generally is less than the number of antigenicepitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate DR4-specific antibodies include: a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 35 to about 92 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 114 to about160 in SEQ ID NO:2; a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 169 to about 240 in SEQ IDNO:2; a polypeptide comprising, or alternatively consisting of, aminoacid residues from about 267 to about 298 in SEQ ID NO:2; a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 330 to about 364 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 391 to about404 in SEQ ID NO:2; and a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 418 to about 465 in SEQ IDNO:2. In this context “about” includes the particularly recited range,larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at eitherterminus or at both termini. As indicated above, the inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe DR4 protein. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. Houghten, R. A., “General method forthe rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986). As oneof skill in the art will appreciate, DR4 polypeptides of the presentinvention and the epitope-bearing fragments thereof described herein(e.g., corresponding to a portion of the extracellular domain such as,for example, amino acid residues 1 to 240 of SEQ ID NO:2) can becombined with parts of the constant domain of immunoglobulins (IgG),resulting in chimeric polypeptides. These fusion proteins facilitatepurification and show an increased half-life in vivo. This has beenshown, e.g., for chimeric proteins consisting of the first two domainsof the human CD4-polypeptide and various domains of the constant regionsof the heavy or light chains of mammalian immunoglobulins (EPA 394,827;Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericDR4 protein or protein fragment alone (Fountoulakis et al., J Biochem270:3958-3964 (1995)).

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

In addition, proteins of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller, M. et al., Nature 310:105-111 (1984)). For example, apeptide corresponding to a fragment of the DR4 polypeptides of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the DR4polypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyricacid, Abu, 2-amino butyric acid, alpha-Abu, alpha-Ahx, 6-amino hexanoicacid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine,norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, alpha-alanine, fluoro-amino acids,designer amino acids such as alpha-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see, e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

The invention additionally, encompasses DR4 polypeptides which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to, specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofDR4 which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivation may be selected from water soluble polymers suchas polyethylene glycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Thepolypeptides may be modified at random positions within the molecule, orat predetermined positions within the molecule and may include one, two,three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

As mentioned, DR4 polypeptides may be modified by either naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques, which are well known in the art. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given DR4 polypeptide.Also, a given DR4 polypeptide may contain many types of modifications.DR4 polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic DR4 polypeptides may result fromnatural posttranslational processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993); POSTRRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

The DR4 polypeptides can be recovered and purified from chemicalsynthesis and recombinant cell cultures by standard methods whichinclude, but are not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxyapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Well-known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and/or purification.

DR4 polynucleotides and polypeptides may be used in accordance with thepresent invention for a variety of applications, particularly those thatmake use of the chemical and biological properties of DR4. Among theseare applications in the treatment and/or prevention of tumors, parasiticinfections, bacterial infections, viral infections, restenosis, andgraft vs. host disease; to induce resistance to parasites, bacteria andviruses; to induce proliferation of T-cells, endothelial cells andcertain hematopoietic cells; to regulate anti-viral responses; and totreat and/or prevent certain autoimmune diseases after stimulation ofDR5 by an agonist. Additional applications relate to diagnosis,treatment, and/or prevention of disorders of cells, tissues andorganisms. These aspects of the invention are discussed further below.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in the cDNAdeposited as ATCC Deposit No. 97853 or encoded by a polynucleotide thathybridizes to the complement of the sequence of SEQ ID NO:1 or containedin the cDNA deposited as ATCC Deposit No. 97853 under stringenthybridization conditions or lower stringency hybridization conditions asdefined supra. The present invention further encompasses polynucleotidesequences encoding an epitope of a polypeptide sequence of the invention(such as, for example, the sequence disclosed in SEQ ID NO:1),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide described herein.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. The term “epitopes,” as usedherein, refers to portions of a polypeptide having antigenic orimmunogenic activity in an animal, preferably a mammal, and mostpreferably in a human. In a preferred embodiment, the present inventionencompasses a polypeptide comprising an epitope, as well as thepolynucleotide encoding this polypeptide. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

Fragments that function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A., “Antibodies That React With PredeterminedSites on Proteins,” Science 219:660-666 (1983). Peptides capable ofeliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate DR4-specific antibodies include: a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 35 to about 92 in FIG. 1 (about 35 to about 92 in SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about 114 to about 160 in FIG. 1 (about 114 to about 160in SEQ ID NO:2); a polypeptide comprising, or alternatively consistingof, amino acid residues from about 169 to about 240 in FIG. 1 (about 169to about 240 in SEQ ID NO:2); a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 267 to about 298 in FIG. 1(about 267 to about 298 in SEQ ID NO:2); a polypeptide comprising, oralternatively consisting of, amino acid residues from about 330 to about364 in FIG. 1 (about 330 to about 364 in SEQ ID NO:2); a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 391 to about 404 in FIG. 1 (about 391 to about 404 in SEQ IDNO:2); and a polypeptide comprising, or alternatively consisting of,amino acid residues from about 418 to about 465 in FIG. 1 (about 418 toabout 465 in SEQ ID NO:2). As indicated above, the inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe DR4 protein.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. Houghten, R. A., “General Method forthe Rapid Solid-Phase Synthesis of Large Numbers of Peptides:Specificity of Antigen-Antibody Interaction at the Level of IndividualAmino Acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986). As oneof skill in the art will appreciate, DR4 polypeptides of the presentinvention and the epitope-bearing fragments thereof described above canbe combined with parts of the constant domain of immunoglobulins (IgG),resulting in chimeric polypeptides. These fusion proteins facilitatepurification and show an increased half-life in vivo. This has beenshown, e.g., for chimeric proteins consisting of the first two domainsof the human CD4-polypeptide and various domains of the constant regionsof the heavy or light chains of mammalian immunoglobulins (EPA 394,827;Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericDR5 protein or protein fragment alone (Fountoulakis et al., J. Biochem.270:3958-3964 (1995)).

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which immunospecifically bind a polypeptide, preferablyan epitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera V_(L) or V_(H) domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. Antibodies that do not bind polypeptides with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies that bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻²M, 10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M,10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M,5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M,5×10¹⁵M, and 10⁻¹⁵M.

Antibodies that bind DR4 receptor polypeptides may bind them as isolatedpolypeptides or in their naturally occurring state. By “isolatedpolypeptide” is intended a polypeptide removed from its nativeenvironment. Thus, a polypeptide produced and/or contained within arecombinant host cell is considered isolated for purposes of the presentinvention. Also intended as an “isolated polypeptide” are polypeptidesthat have been purified, partially or substantially, from a recombinanthost cell. For example, a recombinantly produced version of the DR4polypeptide is substantially purified by the one-step method describedin Smith and Johnson, Gene 67:31-40 (1988). Thus, antibodies of thepresent invention may bind recombinantly produced DR4 receptorpolypeptides.

In a specific embodiment, antibodies of the present invention bind afull-length DR4 receptor expressed on the surface of a cell comprising apolynucleotide encoding amino acids 1 to 468 of SEQ ID NO:2 operablyassociated with a regulatory sequence that controls gene expression. Inanother specific embodiment, antibodies of the present invention bind afull-length DR4 receptor expressed on the surface of a cell comprising apolynucleotide encoding the amino acid sequence encoded by the cDNAcontained in ATCC Deposit No. 97853, operably associated with aregulatory sequence that controls gene expression.

In preferred embodiments, antibodies of the present invention bind themature DR4 receptor expressed on the surface of a cell comprising apolynucleotide encoding amino acids about 24 to about 468 of SEQ ID NO:2operably associated with a regulatory sequence that controls geneexpression. In other preferred embodiments, antibodies of the presentinvention bind mature DR4 receptor expressed on the surface of a cellcomprising a polynucleotide encoding the amino acid sequence of themature polypeptide encoded by the cDNA contained in ATCC Deposit No.97853, operably associated with a regulatory sequence that controls geneexpression.

In preferred embodiments, antibodies of the present invention bind theextracellular domain of a DR4 receptor expressed on the surface of acell comprising a polynucleotide encoding amino acids about 24 to about238 of SEQ ID NO:2 operably associated with a regulatory sequence thatcontrols gene expression. In other preferred embodiments, antibodies ofthe present invention bind the extracellular domain of a DR4 receptorexpressed on the surface of a cell comprising a polynucleotide encodingthe amino acid sequence of the extracellular domain of the polypeptideencoded by the cDNA contained in ATCC Deposit No. 97853, operablyassociated with a regulatory sequence that controls gene expression.

The present invention also provides antibodies that bind DR5polypeptides that act as either DR4 agonists or DR4 antagonists. Inspecific embodiments, the antibodies of the invention stimulateapoptosis of DR4 expressing cells. In other specific embodiments, theantibodies of the invention inhibit TRAIL binding to DR4. In otherspecific embodiments, the antibodies of the invention upregulate DR4expression.

The present invention also provides antibodies that inhibit apoptosis ofDR4 expressing cells. In other specific embodiments, the antibodies ofthe invention downregulate DR4 expression.

In further embodiments, the antibodies of the invention have adissociation constant (K_(D)) of 10⁻⁷ M or less. In preferredembodiments, the antibodies of the invention have a dissociationconstant (K_(D)) of 10⁻⁹ M or less.

The present invention further provides antibodies that stimulateapoptosis of DR4 expressing cells better than an equal concentration ofTRAIL polypeptide stimulates apoptosis of DR4 expressing cells.

The present invention further provides antibodies that stimulateapoptosis of DR4 expressing cells equally well in the presence orabsence of antibody cross-linking reagents; and/or stimulate apoptosiswith equal or greater potency as an equal concentration of TRAIL in theabsence of a cross-linking antibody or other cross-linking agent.

In further embodiments, antibodies of the invention have an off rate(k_(off)) of 10⁻³/sec or less. In preferred embodiments, antibodies ofthe invention have an off rate (k_(off)) of 10⁻⁴/sec or less. In otherpreferred embodiments, antibodies of the invention have an off rate(k_(off)) of 10⁻⁵/sec or less.

The present invention further encompasses methods and compositions forkilling of cells expressing DR4 on their surface, comprising, oralternatively consisting of, contacting anti-DR4 antibodies of theinvention with such cells expressing DR4 on their surface.

In specific embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, contacting anti-DR4antibodies of the invention with such cells expressing DR4 on theirsurface.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising amino acids about 52 to about 184 of SEQ ID NO:2on their surface, comprising, or alternatively consisting of, contactinganti-DR4 antibodies of the invention with such cells expressing saidpolypeptide on their surface.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising the extracellular domain of the polypeptideencoded by the cDNA clone contained in ATCC Deposit No. 97920 on theirsurface, comprising, or alternatively consisting of, contacting anti-DR4antibodies of the invention with such cells expressing said polypeptideon their surface.

The present invention further encompasses methods and compositions forkilling of cells expressing DR4 on their surface, comprising, oralternatively consisting of, administering to an animal, anti-DR4antibodies of the invention in an amount effective to kill such DR4expressing cells.

In specific embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, administering to ananimal, anti-DR4 antibodies of the invention in an amount effective toinduce apoptosis in such DR4 expressing cells.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising amino acids about 52 to about 184 of SEQ ID NO:2on their surface, comprising, or alternatively consisting of,administering to an animal, anti-DR4 antibodies of the invention in anamount effective to induce apoptosis in such cells expressing saidpolypeptide on their surface.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising the extracellular domain of the polypeptideencoded by the cDNA clone contained in ATCC Deposit No. 97920 on theirsurface, comprising, or alternatively consisting of, administering to ananimal, anti-DR4 antibodies of the invention in an amount effective toinduce apoptosis in such cells expressing said polypeptide on theirsurface.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand or receptor activity by at least 90%,at least 80%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. Thus, the inventionfurther relates to antibodies which act as agonists or antagonists ofthe polypeptides of the present invention. The above antibody agonistscan be made using methods known in the art. See, e.g., PCT publicationWO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988(1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al.,J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res.58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179(1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard etal., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al.,Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem.272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Example 29, below, as well as Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)(incorporated by reference herein in its entirety).

Furthermore, antibodies of the present invention may be used to causedeath of cells which express polypeptides of the present invention,including both in vitro and in vivo diagnostic and therapeutic methods.For example, the antibodies have use in immunoassays for qualitativelyand quantitatively measuring levels of the polypeptides of the presentinvention in biological samples. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)(incorporated by reference herein in its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, or toxins. See, e.g., PCT publicationsWO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen of interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. Monoclonal antibodies can be prepared using a widevariety of techniques known in the art including the use of hybridomaand recombinant and phage display technology.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well-known in the art and arediscussed in detail in Example 21. Briefly, mice can be immunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B-cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J 7(5):437-444(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Antibodies of the invention may also include multimeric forms ofantibodies. For example, antibodies of the invention may take the formof antibody dimers, trimers, or higher-order multimers of monomericimmunoglobulin molecules. Dimers of whole immunoglobulin molecules or ofF(ab′)₂ fragments are tetravalent, whereas dimers of Fab fragments orscFv molecules are bivalent. Individual monomers within an antibodymultimer may be identical or different, i.e., they may be heteromeric orhomomeric antibody multimers. For example, individual antibodies withina multimer may have the same or different binding specificities.

Multimerization of antibodies may be accomplished through naturalaggregation of antibodies or through chemical or recombinant linkingtechniques known in the art. For example, some percentage of purifiedantibody preparations (e.g., purified IgG1 molecules) spontaneously formprotein aggregates containing antibody homodimers, and otherhigher-order antibody multimers. Alternatively, antibody homodimers maybe formed through chemical linkage techniques known in the art. Forexample, heterobifunctional crosslinking agents including, but notlimited to, SMCC [succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidylS-acethylthio-acetate] (available, for example, from PierceBiotechnology, Inc. (Rockford, Ill.)) can be used to form antibodymultimers. An exemplary protocol for the formation of antibodyhomodimers is given in Ghetie et al., Proceedings of the NationalAcademy of Sciences USA (1997) 94:7509-7514, which is herebyincorporated by reference in its entirety. Antibody homodimers can beconverted to Fab′2 homodimers through digestion with pepsin.Alternatively, antibodies can be made to multimerize through recombinantDNA techniques. IgM and IgA naturally form antibody multimers throughthe interaction with the J chain polypeptide. Non-IgA or non-IgMmolecules, such as IgG molecules, can be engineered to contain the Jchain interaction domain of IgA or IgM, thereby conferring the abilityto form higher order multimers on the non-IgA or non-IgM molecules.(see, for example, Chintalacharuvu et al., (2001) Clinical Immunology101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94.,both of which are hereby incorporated by reference in their entireties.)ScFv dimers can also be formed through recombinant techniques known inthe art; an example of the construction of scFv dimers is given in Goelet al., (2000) Cancer Research 60:6964-6971, which is herebyincorporated by reference in its entirety. Antibody multimers may bepurified using any suitable method known in the art, including, but notlimited to, size exclusion chromatography.

A. Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source (e.g., an antibodycDNA library, or a cDNA library generated from, or nucleic acid,preferably poly A+ RNA, isolated from, any tissue or cells expressingthe antibody, such as hybridoma cells selected to express an antibody ofthe invention) by PCR amplification using synthetic primers hybridizableto the 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-554) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,1988, Science 242:1038-1041).

B. Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, e.g., a heavy or light chain of anantibody of the invention, requires construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., 1987, Methods inEnzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro,which confers resistance to hygromycin (Santerre et al., 1984, Gene30:147). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplif cation for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

C. Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. Also encompassed are antibodies of theinvention recombinantly fused or chemically conjugated (including bothcovalently and non-covalently conjugations) to a polypeptide (or portionthereof, preferably at least 10, 20 or 50 amino acids of thepolypeptide) to generate fusion proteins. The fusion does notnecessarily need to be direct, but may occur through linker sequences.The antibodies may be specific for antigens other than polypeptides (orportion thereof, preferably at least 10, 20 or 50 amino acids of thepolypeptide) of the present invention. Furthermore, the antibodies maybe specific for polypeptides (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention.For example, antibodies may be used to target the polypeptides of thepresent invention to particular cell types, either in vitro or in vivo,by fusing or conjugating the polypeptides of the present invention toantibodies specific for particular cell surface receptors.Alternatively, antibodies of the present invention may be used to targetconjugated polypeptides and/or compounds to particular cell types,either in vitro or in vivo, by fusing or conjugating the antibodies ofthe present invention to the polypeptides and/or compounds to betargeted.

Antibodies fused or conjugated to the polypeptides of the presentinvention may also be used in in vitro immunoassays and purificationmethods using methods known in the art. Also, antibodies of the presentinvention fused or conjugated to polypeptides and/or compounds may beused in in vitro immunoassays and purification methods using methodsknown in the art. See e.g., Harbor et al., supra, and PCT publication WO93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994);U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fellet al., J. Immunol. 146:2446-2452 (1991), which are incorporated byreference in their entireties.

The present invention further includes compositions comprising thepolypeptides, including antibodies, of the present invention fused orconjugated to antibody domains other than the variable regions.Furthermore, the present invention includes compositions comprising theantibodies of the present invention fused or conjugated to heterologousantibody domains other than variable regions. For example, thepolypeptides including antibodies of the present invention may be fusedor conjugated to a heterologous antibody Fc region, or portion thereof.

The antibody portion fused to a polypeptide and/or antibody of thepresent invention may comprise the constant region, hinge region, CH1domain, CH2 domain, and CH3 domain or any combination of whole domainsor portions thereof. The polypeptides, including antibodies, may also befused or conjugated to the above antibody portions to form multimers.For example, Fc portions fused to the polypeptides, includingantibodies, of the present invention can form dimers through disulfidebonding between the Fc portions. Higher multimeric forms can be made byfusing the polypeptides, including antibodies of the present invention,to portions of IgA and IgM. Methods for fusing or conjugating thepolypeptides, including antibodies, of the present invention to antibodyportions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603;5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al.,Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci.USA 89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides, including antibodies, of thepresent invention may be fused or conjugated to the above antibodyportions to increase the in vivo half life of the polypeptides or foruse in immunoassays using methods known in the art. Further, thepolypeptides, including antibodies, of the present invention may befused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84-86 (1988).

The polypeptides, including antibodies, of the present invention fusedor conjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizing,agonizing and/or antagonizing other molecules, than the monomericsecreted antibody, protein, antibody fragment or protein fragment alone.(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases,the heterologous Fc part in a fusion protein is beneficial in therapyand diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5 receptor, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).

The present invention further includes compositions comprising theantibodies of the present invention fused or conjugated to human serumalbumin to increase the in vivo half-life of the antibodies or for usein immunoassays using methods known in the art. Further, the antibodiesof the present invention may be fused or conjugated to human serumalbumin to facilitate purification. In many cases, the human serumalbumin part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokineticproperties. See e.g., U.S. Pat. No. 5,876,969, EP Patent 0413622, andU.S. Pat. No. 5,766,883, herein incorporated by reference in theirentirety.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitates theirpurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment and/or prevention regimens.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 formetal ions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, 3-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and acquorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramnicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Additionally, antibodies of the invention may be modified bypost-translational modifications encompassed including, for example,N-linked or O-linked carbohydrate chains, processing of N-terminal orC-terminal ends, attachment of chemical moieties to the amino acidbackbone, chemical modifications of N-linked or O-linked carbohydratechains, and addition or deletion of an N-terminal methionine residue asa result of procaryotic host cell expression.

Also provided by the invention are chemically modified antibodyderivatives, which may provide additional advantages such as increasedsolubility, stability and circulating time of the antibody, or decreasedimmunogenicity (see, U.S. Pat. No. 4,179,337). The chemical moieties forderivation may be selected from water soluble polymers such aspolyethylene glycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Theantibodies may be modified at random positions within the molecule, orat predetermined positions within the molecule and may include one, two,three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the antibody with consideration of effects on bindingspecificity and agonistic and/or antagonistic properties of theantibody.

As described supra, there are a number of attachment methods availableto those skilled in the art, e.g., EP 0 401 384, herein incorporated byreference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresylchloride) and polyethylene glycol may be attached to antibodies vialinkage to any of a number of amino acid residues. Furthermore, one mayspecifically desire antibodies chemically modified at the N-terminus.

Polyethylene glycol may be attached to the antibody either directly orby an intervening linker. Linkerless systems for attaching polyethyleneglycol to proteins are described in Delgado et al., Crit. Rev. Thera.Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J ofHematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No.5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each ofwhich are incorporated herein by reference.

As described supra, polyethylene glycol can also be attached toantibodies using a number of different intervening linkers. See e.g.,U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporatedherein by reference.

The number of polyethylene glycol moieties attached to each antibody ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated antibodies of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5,4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per antibody molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

As described supra, antibodies may be modified by natural processes,such as posttranslational processing, or by chemical modificationtechniques, which are well known in the art. It will be appreciated thatthe same type of modification may be present in the same or varyingdegrees at several sites in a given antibody. Also, a given antibody maycontain many types of modifications.

Modifications may include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

D. Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP40 or Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate atpH 7.2, 1% Trayslol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody.

E. Antibody Based Therapies

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingand/or preventing one or more of the disorders or conditions describedherein. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof as described herein).

While not intending to be bound to theory, DR4 receptors are believed toinduce programmed cell death by a process which involves theassociation/cross-linking of death domains between different receptormolecules. Further, DR4 ligands (e.g., TRAIL) which induce DR4 mediatedprogrammed cell death are believed to function by causing theassociation/cross-linking of DR4 death domains. Thus, agents (e.g.,antibodies) which prevent association/cross-linking of DR4 death domainswill prevent DR4 mediated programmed cell death, and agents (e.g.,antibodies) which facilitate the association/cross-linking of DR4 deathdomains will induce DR4 mediated programmed cell death.

As noted above, DR4 receptors have been shown to bind TRAIL. DR4receptors are also known to be present in a number of tissues and on thesurfaces of a number of cell types. These tissues and cell types includeamniotic cells, heart, liver cancer cells, kidney, leukocytes, activatedT-cells, K562 cells (an erythroleukemia cell line) plus PMA, W138 cells(a human lung fibroblast cell line), Th2 cells (lymphocytes), humantonsils, and CD34 depleted buffy coat cells of cord blood. Further, asexplained in more detail below, TRAIL has been shown to induce apoptosisand to inhibit the growth of tumor cells in vivo. Additionally, TRAILactivities are believed to be modulated, at least in part, throughinteraction with DR4 and DR5 receptors.

TRAIL is a member of the TNF family of cytokines which has been shown toinduce apoptotic cell death in a number of tumor cell lines and appearsto mediate its apoptosis inducing effects through interaction with DR4and DR5 receptors. These death domain containing receptors are believedto form membrane-bound self-activating signaling complexes whichinitiate apoptosis through cleavage of caspases.

In addition to DR4 and DR5 receptors, TRAIL also binds to severalreceptors proposed to be “decoy” receptors, DcR2 (a receptor with atruncated death domain), DcR1 (a GP1-anchored receptor), and OPG (asecreted protein which binds to another member of the TNF family,RANKL).

Further, recent studies have shown that the rank-order of affinities ofTRAIL for the recombinant soluble forms of its receptors is stronglytemperature dependent. In particular, at 37° C., DR5 has the highestaffinity for TRAIL and OPG having the lowest affinity.

The DR4 and DR5 receptor genes, as well as genes encoding two decoyreceptors, have been shown to be located on human chromosome 8p21-22.Further, this region of the human genome is frequently disrupted in headand neck cancers.

It has recently been found that the FaDu nasopharyngeal cancer cell linecontains an abnormal chromosome 8p21-22 region. (Ozoren et al., Int. J.Oncol. 16:917-925 (2000).) In particular, a homozygous deletioninvolving DR4, but not DR5, has been found in these cells. (Ozoren etal., Int. J. Oncol. 16:917-925 (2000).) The homozygous loss within theDR4 receptor gene in these FaDu cells encompasses the DR4 receptor deathdomain. This disruption of the DR4 receptor death domain is associatedwith resistance to TRAIL-mediated cytotoxicity. Further, re-introductionof a wild-type DR4 receptor gene has been shown to both lead toapoptosis and restoration of TRAIL sensitivity of FaDu cells. (Ozoren etal., Int. J. Oncol. 16:917-925 (2000).) These data indicate that the DR4receptor gene may be inactivated in human cancers and DR4 receptor genedisruption may contribute to resistance to TRAIL therapy. It is expectedthat similar results would be found in cells having analogous deletionsin the DR5 gene.

It has also been shown that overexpression of the cytoplasmic domain ofthe DR4 receptor in human breast, lung, and colon cancer cell linesleads to p53-independent apoptotic cell death which involves thecleavage of caspases. (Xu et al., Biochem. Biophys. Res. Commun.269:179-190 (2000).) Further, DR4 cytoplasmic domain overexpression hasalso been shown to result in cleavage of both poly(ADP-ribose)polymerase (PARP) and a DNA fragmentation factor (i.e., ICAD-DFF45). (Xuet al., Biochem. Biophys. Res. Commun. 269:179-190 (2000).) In addition,despite similar levels of DR4 cytoplasmic domain protein as compared tocancer cells tested, normal lung fibroblasts have been shown to beresistant to DR4 cytoplasmic domain overexpression and show no evidenceof caspase-cleavage. (Xu et al., Biochem. Biophys. Res. Commun.269:179-190 (2000).) Again, similar results are expected with cells thatoverexpress the cytoplasmic domain of DR5. Thus, the cytoplasmic domainsof the DR4 and DR5 receptors are useful as agents for inducingapoptosis, for example, in cancer cells.

Further, overexpression of the cyclin-dependent kinase inhibitorp21(WAF1/CIP1), as well as the N-terminal 91 amino acids of thisprotein, has cell cycle-inhibitory activity and inhibits DR4 cytoplasmicdomain-dependent caspase cleavage. Thus, DR4 receptors are also involvedin the regulation of cell cycle progression. As above, similar resultsare expected with the DR5 receptor. Thus, the DR4 and DR5 receptors, aswell as agonists and antagonists of these receptors, are useful forregulating cell cycle progression.

Antibodies which bind to DR4 receptors are useful for treating and/orpreventing diseases and conditions associated with increased ordecreased DR4-induced apoptotic cells death. Further, these antibodiesvary in the effect they have on DR4 receptors. These effects differbased on the specific portions of the DR4 receptor to which theantibodies bind, the three-dimensional conformation of the antibodymolecules themselves, and/or the manner in which they interact with theDR4 receptor. Thus, antibodies which bind to the extracellular domain ofa DR4 receptor can either stimulate or inhibit DR4 activities (e.g., theinduction of apoptosis). Antibodies which stimulate DR4 receptoractivities (e.g., by facilitating the association between DR4 receptordeath domains) are DR4 agonists, and antibodies which inhibit DR4receptor activities (e.g., by blocking the binding of TRAIL and/orpreventing the association between DR4 receptor death domains) are DR4antagonists.

Antibodies of the invention which function as agonists and antagonistsof DR4 receptors include antigen-binding antibody fragments such as Faband F(ab′)₂ fragments, Fd, single-chain Fvs (scFv), disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain, as wellas polyclonal, monoclonal and humanized antibodies. Divalent antibodiesare preferred as agonists. Each of these antigen-binding antibodyfragments and antibodies are described in more detail elsewhere herein.

In view of the above, antibodies of the invention, as well as otheragonists, are useful for stimulating DR4 death domain activity topromote apoptosis in cells which express DR4 receptors (e.g., cancercells). Antibodies of this type are useful for prevention and/ortreating diseases and conditions associated with increased cell survivaland/or insensitivity to apoptosis-inducing agents (e.g., TRAIL), such assolid tissue cancers (e.g., skin cancer, head and neck tumors, breasttumors, endothelioma, lung cancer, osteoblastoma, osteoclastoma, andKaposi's sarcoma) and leukemias.

Antagonists of the invention (e.g., anti-DR4 antibodies) function bypreventing DR4 mediated apoptosis and are useful for preventing and/ortreating diseases associated with increased apoptotic cell death.Examples of such diseases include diabetes mellitus, AIDS,neurodegenerative disorders, myelodysplastic syndromes, ischemic injury,toxin-induced liver disease, septic shock, cachexia and anorexia.

As noted above, DR4 receptors are present on the surfaces of T-cells.Thus, agonists of the invention (e.g., anti-DR4 receptor antibodies) arealso useful for inhibiting T-cell mediated immune responses, as well aspreventing and/or treating diseases and conditions associated withincreased T-cell proliferation. Diseases and conditions associated withT-cell mediated immune responses and increased T-cell proliferationinclude graft-v-host responses and diseases, osteoarthritis, psoriasis,septicemia, inflammatory bowel disease, inflammation in general,autoimmune diseases, and T-cell leukemias.

When an agonist of the invention is administered to an individual forthe treatment and/or prevention of a disease or condition associatedwith increased T-cell populations or increased cell proliferation (e.g.,cancer), the antagonist may be co-administered with another agent whichinduces apoptosis (e.g., TRAIL) or otherwise inhibits cell proliferation(e.g., an anti-cancer drug). Combination therapies of this nature, aswell as other combination therapies, are discussed below in more detail.

Further, antagonists of the invention (e.g., anti-DR4 receptorantibodies) are also useful for enhancing T-cell mediated immuneresponses, as well as preventing and/or treating diseases and conditionsassociated with decreased T-cell proliferation. Antibodies of theinvention which block the binding of DR4 receptor ligands to DR4receptors or interfere with DR4 receptor conformational changesassociated with membrane signal transduction can inhibit DR4 mediatedT-cell apoptosis. The inhibition of DR4-mediated apoptosis can, forexamples, either result in an increase in the expansion rate of in vivoT-cell populations or prevent a decrease in the size of suchpopulations. Thus, antagonists of the invention can be used to preventand/or treat diseases or conditions associated with decreased ordecreases in T-cell populations. Examples of such diseases andconditions included acquired immune deficiency syndrome (AIDS) andrelated afflictions (e.g., AIDS related complexes), T-cellimmunodeficiencies, radiation sickness, and T-cell depletion due toradiation and/or chemotherapy.

When an antagonist of the invention is administered to an individual forthe treatment and/or prevention of a disease or condition associatedwith decreased T-cell populations, the antagonist may be co-administeredwith an agent which activates and/or induces lymphocyte proliferation(e.g., a cytokine). Combination therapies of this nature, as well asother combination therapies, are discussed below in more detail.

Similarly, agonists and antagonists of the invention (e.g., anti-DR5receptor antibodies) are also useful when administered alone or incombination with another therapeutic agent for either inhibiting orenhancing B-cell mediated immune responses, as well as preventing and/ortreating diseases and conditions associated with increased or decreasedB-cell proliferation.

Anti-DR4 antibodies are thus useful for treating and/or preventingmalignancies, premalignant conditions, abnormalities, diseases and/orconditions involving tissues and cell types which express DR4 receptors.Further, malignancies, premalignant conditions, abnormalities, diseasesand/or conditions which can be treated and/or prevented by the inductionof programmed cell death in cells which express DR4 receptors can betreated and/or prevented using DR4 receptor agonists of the invention.Similarly, malignancies, premalignant conditions, abnormalities,diseases and/or conditions which can be treated and/or prevented byinhibiting programmed cell death in cells which express DR4 receptorscan be treated and/or prevented using DR4 receptor antagonists of theinvention.

A number of additional malignancies, premaligant conditions,abnormalities, diseases and/or conditions which can be treated using theagonists and antagonists of the invention are set out elsewhere herein,for example, in the section below entitled “Therapeutics”.

The antibodies of the present invention may be used therapeutically in anumber of ways. For example, antibodies which bind polynucleotides orpolypeptides of the present invention can be administered to anindividual (e.g., a human) either locally or systemically. Further,these antibodies can be administered alone, in combination with anothertherapeutic agent, or associated with or bound to a toxin.

The present invention provides antibodies, which may be administered incombination with one or more therapeutic agents and/or procedures in thetreatment, prevention, amelioration and/or cure of cancers. In preferredembodiments, agonistic antibodies of the invention may be administeredin combination with one or more therapeutic agents and/or procedures inthe treatment, prevention, amelioration and/or cure of cancers andpremalignant conditions.

In a specific embodiment, antibodies or antibody compositions of theinvention are administered in combination with DAB₃₈₉EGF, a diphtheriatoxin fused to Epidermal Growth Factor. DAB₃₈₉EGF is described in Shawet al., (1991) The Journal of Biological Chemistry, 266:21118-24, whichis hereby incorporated by reference in its entirety. In a specificembodiment, antibodies or antibody compositions of the invention areadministered in combination with DAB₃₈₉EGF for the treatment of cancer,such as brain cancers and epithelial cancers. In a specific embodiment,antibodies or antibody compositions of the invention are administered incombination with DAB₃₈₉EGF for the treatment of astrocytomas. In aspecific embodiment, antibodies or antibody compositions of theinvention are administered in combination with DAB₃₈₉EGF for thetreatment of glioblastyoma multiforme (GBM).

Therapeutic agents, useful in the treatment, prevention, ameliorationand/or cure of cancers and premalignant conditions, with whichantibodies of the present invention may be administered, include, forexample, biological agents (e.g., inhibitors of signaling pathways,inhibitors of gene transcription, inhibitors of multi-drug resistance(MDR) mechanisms, inhibitors of angiogenesis, inhibitors of matrixmetalloproteinases, hormones and hormone antagonists, and compounds ofunknown mechanism), chemotherapeutic agents (e.g., alkylating agents,antimetabolites, farnesyl transferase inhibitors, mitotic spindleinhibitors (plant-derived alkaloids), nucleotide analogs, platinumanalogs, and topoisomerase inhibitors), corticosteroids, gene therapies,immunotherapeutic agents (e.g., monoclonal antibodies, cytokines andvaccines), phototherapy, radiosensitizing agents, treatment supportagents (e.g., anti-emetic agents, analgesic agents and hematopoieticagents), and other miscellaneous drug types. Therapeutic procedures,useful in the treatment, prevention, amelioration and/or cure of cancersand premalignant conditions, with which agonistic antibodies of thepresent invention may be administered, include, for example, but are notlimited to, surgical procedures and radiation therapies.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, prevention, amelioration and/orcure of cancers and premalignant conditions.

In specific embodiments, antibodies of the present invention may beadministered in combination with one or more therapeutic agents usefulin the treatment, prevention, amelioration and/or cure of cancers andpremalignant conditions including, but not limited to, 81C6(Anti-tenascin monoclonal antibody), 2-chlorodeoxyadenosine, A007(4-4′-dihydroxybenzophenone-2, 4-dinitrophenylhydrazone), Abarelixg(Abarelix-Depot-Mg, PPI-149, R-3827); Abiraterone acetates (CB-7598,CB-7630), ABT-627 (ET-1 inhibitor), ABX-EGF (anti-EGFr MAb),Acetyldinaline (CI-994, GOE-5549, GOR-5549, PD-130636), AG-2034(AG-2024, AG-2032, GARFT [glycinamide ribonucleoside transformylase]inhibitor), Alanosine, Aldesleukin (IL-2, Proleukin®), Alemtuzumab®(Campath®), Alitretinoin (Panretin®, LGN-1057), Allopurinol (Aloprim®D,Zyloprim®), Altretamine (Hexylen®, hexamethylmelamine, Hexastat®),Amifostine (Ethyol®), Aminocamnptothecin (9-AC, 9-Aminocamptothecin, NSC603071), Aminoglutethimide (Cytadren®), Aminolevulinic acid (Levulan®,Kerastick®), Aminopterin, Amsacrine, Anastrozole (Arimidex®),Angiostatin, Annamycin (AR-522, annamycin LF, Aronex®), Anti-idiotypetherapy (BsAb), Anti-CD 19/CD3 MAb (anti-CD 19/CD3 scFv, anti-NHL MAb),APC-8015 (Provenge®, Dendritic cell therapy), Aplidine (Aplidin®,Aplidina®), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®, Compound506U78), Arsenic trioxide (Trisenox®, ATO, Atrivex®), Avorelin®(Meterelin®, MF-6001, EP-23904), B43-Genistein (anti-CD19 Ab/genisteinconjugate), B43-PAP (anti-CD19 Ab/pokeweed antiviral protein conjugate),B7 antibody conjugates, BAY 43-9006 (Raf kinase inhibitor), BBR 3464,Betathine (Beta-LT), Bevacizumab® (Anti-VEGF monoclonal antibody,rhuMAb-VEGF), Bexarotene (Targreting, LGD1069), BIBH-1 (Anti-FAP MAb),BIBX-1382, Biclutamide (Casodex®), Biricodar dicitrate (Incel®, IncelMDR Inhibitor), Bleomycin (Blenoxane®), BLP-25 (MUC-1 peptide), BLySantagonists, BMS-214662 (BMS-192331, BMS-193269, BMS-206635), BNP-1350(BNPI-1100, Karenitecins), Boronated Protoporphyrin Compound (PDIT,Photodynarnic Immunotherapy), Bryostatin-1 (Bryostatin®g, BMY-45618,NSC-339555), Budesonide (Rhinocort®), Busulfan (Busulfex®, Myleran®),C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb, Cetuximab®), C242-DM1(huC242-DM1), Cabergoline (Dostinex®), Capecitabine (Xeloda®,Doxifluridine®, oral 5-FU), Carbendazin® (FB-642), Carboplatin(Paraplatin®, CBDCA), Carboxyamidotriazole (NSC 609974, CAI, L-651582),Cannustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), CC49-zeta genetherapy, CEA-cide® (Labetuzumab®, Anti-CEA monoclonal antibody, hMN-14),CeaVac® (MAb 3H1), Celecoxib (Celebrex®), CEP-701 (KT-5555), Cereport®(Lobradimil®, RMP-7), Chlorambucil (Leukeran®), CHML (CytotropicHeterogeneous Molecular Lipids), Cholecaliferol, CI-1033 (Pan-erbB RTKinhibitor), Cilengitide (EMD-121974, integrin alphavbeta3 antagonist),Cisplatin (Platinol®, CDDP), Cisplatin-epinephrine gel (IntraDose®,FocaCist®), Cisplatin-liposomal (SPI-077), 9-cis retinoic acid (9-cRA),Cladribine (2-CdA, Leustatin®), Clofarabine (chloro-fluoro-araA),Clonadine hydrochloride (Duraclon®), CMB-401 (Anti-PEMMAb/calicheamycin), CMT-3 (COL-3, Metastat®), Cordycepin, Cotara®(chTNT-1/B, [¹³¹I]-chTNT-1/B), CN-706, CP-358774 (Tarceva®, OSI-774,EGFR inhibitor), CP-609754, CP IL-4-toxin (IL-4 fusion toxin), CS-682,CT-2584 (Apra®, CT-2583, CT-2586, CT-3536), CTP-37 (Avicine®, hCGblocking vaccine), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine(Cytosar-US, ara-C, cytosine arabinoside, DepoCyte), D-limonene,DAB389-EGF (EGF fusion toxin), Dacarbazine (DTIC), Daclizumab®(Zenapax®), Dactinomycin (Cosmegen®), Daunomycin (Daunorubicin®,Cerubidine®), Daunorubicin (DaunoXome®, Daunorubicin®, Cerubidine®),DeaVac® (CEA anti-idiotype vaccine), Decitabine (5-aza-2′-deoxyytidine),Declopramide (Oxi-104), Denileukin diftitox (Ontak®), Depsipeptide(FR901228, FK228), Dexamethasone (Decadron®), Dexrazoxane (Zinecard®),Diethylnorspennine (DENSPM), Diethylstilbestrol (DES),Dihydro-5-azacytidine, Docetaxel (Taxotere®, Taxane®), Dolasetronmesylate (Anzemet®), Dolastatin-10 (DOLA-10, NSC-376128), Doxorubicin(Adriamycin®, Doxil®, Rubex®), DPPE, DX-8951f (DX-8951), Edatrexate,EGF-P64k Vaccine, Elliott's B Solution®, EMD-121974, Endostatin,Eniluracil (776c85), EO9 (EO1, EO4, EO68, EO70, EO72), Epirubicin(Ellence®, EPI, 4′ epi-doxorubicin), Epratuzumab® (Lymphocide®,humanized anti-CD22, HAT), Erythropoietin (EPO®, Epogen®), Procrit®),Estramustine (Emcyt®), Etanidazole (Radinyl®), Etoposide phosphate(Etopophos®), Etoposide (VP-16, Vepesid®), Exemestane (Aromasin®,Nikidess®), Exetecan mesylate (DX-8951, DX-8951f), Exisulind (SAAND,Aptosyn®, cGMP-PDE2 and 5 inhibitor), F19 (Anti-FAP monoclonal antibody,iodinated anti-FAP MAb), Fadrozole (Afema®, Fadrozole hydrochloride,Arensin®), Fenretinideg (4HPR), Fentanyl citrate (Actiq®), Filgrastim(Neupogen®, G-CSF), FK-317 (FR-157471, FR-70496), Flavopiridol(HMR-1275), Fly3/flk2 ligand (Mobista®), Fluasterone, Fludarabine(Fludara®, FAMP), Fludeoxyglucose (F-18®), Fluorouracil (5-FU, Adrucil®,Fluoroplex®, Efudex®), Flutamide (Eulexin®), FMdC (KW-2331, MDL-101731),Formestane (Lentaron®), Fotemustine (Muphoran®, Mustophoran®), FUDR(Floxuridine®), Fulvestrant (Faslodex®), G3139 (Genasense®,GentaAnticode®, Bcl-2 antisense), Gadolinium texaphyrin (Motexafingadolinium, Gd-Tex®, Xcytrin®), Galarubicin hydrochloride (DA-125),GBC-590, Gastrimmune® (Anti-gastrin-17 immunogen, anti-g17), Gemcitabine(Gemto®, Gemzar®), Gentuzumab-ozogamicin (Mylotarg®), GL331, Globo Hhexasaccharide (Globo H-KLH®), Glufosfamide®& (O-D-glucosyl-isofosfamidemustard, D19575, INN), Goserelin acetate (Zoladex®)), Granisetrori(Kytril™)), GVAX (GM-CSF gene therapy), Her-2/Neu vaccine, Herceptin®(Trastuzumab®, Anti-HER-2 monoclonal antibody, Anti-EGFR-2 MAb),HSPPC-96 (HSP cancer vaccine, gp96 heat shock protein-peptide complex),HulDiO (anti-HLA-DR MAb, SMART ID10), HumaLYM (anti-CD20 MAb),Hydrocortisone, Hydroxyurea (Hydrea®), Hypericing (VIMRxyn®), 1-131Lipidiol®, Ibritumomab® tiuxetan (Zevalin®), Idarubicin (Idamycin®,DMDR, IDA), Ifosfamide (IFEX®), Imatinib mesylate (STI-571, Imatinib®,Glivece, Gleevec®, Ab1 tyrosine kinase inhibitor), INGN-101 (p53 genetherapy/retrovirus), INGN-201 (p53 gene therapy/adenovirus), Interferonalpha (Alfaferone®, Alpha-IF®), Interferon alpha 2a (Intron A®),Interferon gamma (Gamma-interferon, Gamma 1000, Gamma-IF), Interleukin-2(ProleiukinR®), Intoplicine (RP 60475), Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1), Irofulven (MGI-114, Ivofulvan, Acylfulveneanalogue), ISIS-2053 (PKC-alpha antisense), ISIS-2503 (Ras antisense),ISIS-3521 (PKC-alpha antisense), ISIS-5132 (K-ras/raf antisense),Isotretinoin (13-CRA, 13-cis retinoic acid, Accutane®), Ketoconazole(Nizoral®), KRN-8602 (MX, MY-5, NSC-619003, MX-2), L-778123 (Rasinhibitors), L-asparaginase (Elspar®, Crastinin®, Asparaginase Medac®,Kidrolase®), Leflunomide (SU-101, SU-0200), Letrozole (Femara®),Leucovorin (Leucovorin®, Wellcovorin®), Leuprolide acetate (Viadur®,Lupron®, Leuprogel®, Eligard®), Leuvectin® (cytofectin+IL-2 gene, IL-2gene therapy), Levamisole (Ergamisol®), Liarozole (Liazal, Liazol,R-75251, R-85246, Ro-85264), Lmb-2 immunotoxin (anti-CD25 recombinantimmuno toxin, anti-Tac(Fv)-PE38), Lometrexol (T-64, T-904064), Lomustine(CCNU®, CeeNU®), LY-335979, Lym-1 (131-I LYM-1), Lymphoma vaccine(Genitope), Mannan-MUC1 vaccine, Marimastat® (BB-2516, TA-2516, MMPinhibitor), MDX447 (MDX-220, BAB-447, EMD-82633, H447,anti-EGFr/FcGammaR1r), Mechlorethamine (Nitrogen Mustard, HN₂,Mustargen®), Megestrol acetate (Megace®, Pallace®), Melphalan (L-PAM,Alkeran®, Phenylalanine mustard), Mercaptopurine (6-mercaptopurine,6-MP), Mesna (Mesnex®), Methotrexate® (MTX, Mexate®, Folex®),Methoxsalen (Uvadex®), 2-Methoxyestradiol (2-ME, 2-ME2),Methylprednisolone (Solumedrol®), Methyltestosterone (Android-10®,Testred®, Virilon®), MGV, Mitomycin C (Mitomycin®, Mutamycin®, MitoExtra®), Mitoxantrone (Novantrone®, DHAD), Mitumomab® (BEC-2,EMD-60205), Mivobulin isethionate (CI-980), MN-14 (Anti-CEAimmunoradiotherapy, ¹³¹I-MN-14, ¹⁸⁸Re-MN-14), Motexafin Lutetium(Lutrin®, Optrin®, Lu-Tex®, lutetium texaphyrin, Lucyn®, Antrin®),MPV-2213ad (Finrozole®), MS-209, Muc-1 vaccine, NaPro Paclitaxel,Nelarabine (Compound 506, U78), Neovastat® (AE-941, MMP inhibitor),Neugene compounds (Oncomyc-NG, Resten-NG, myc antisense), Nilutamide(Nilandron®), NovoMAb-G2 scFv (NovoMAb-G2 IgM), NPI-0052 (proteasomeinhibitor), O6-benzylguanine (BG, Procept®), Octreotide acetate(Sandostatin LAR® Depot), Odansetron (Zofran®), Onconase (Ranpirnase®),OncoVAX-CL, OncoVAX-CL Jenner (GA-733-2 vaccine), OncoVAX-P(OncoVAX-PrPSA), Onyx-015 (p53 gene therapy), Oprelvekin (Neumageo),Orzel (Tegafur+Uracil+Leucovorin), Oxaliplatin (Eloxatine®, Eloxatin®),Pacis® (BCG, live), Paclitaxel (Paxene®, Taxol®), Paclitaxel-DHA(Taxoprexin®), Pamidronate (Aredia®), PC SPES, Pegademase (Adagen®,Pegademase bovine), Pegaspargase®V (Oncospar®), Peldesine (BCX-34, PNPinhibitor), Pemetrexed disodium (Alimta®, MTA, multitargeted antifolate,LY 231514), Pentostatin (Nipent®, 2-deoxycoformycin), Perfosfamide(4-hydroperoxycyclophosphamide, 4-HC), Perillyl alcohol (perillaalcohol, perillic alcohol, perillol, NSC-641066), Phenylbutyrate,Pirarubicin (THP), Pivaloyloxymethyl butyrate (AN-9, Pivanex®), Porfimersodium (Photofrin®), Prednisone, Prinomastat® (AG-3340, MMP inhibitor),Procarbazine (Matulane®), PROSTVAC, Providence Portland Medical CenterBreast Cancer Vaccine, PS-341 (LDP-341, 26S proteasome inhibitor), PSMAMAb (Prostate Specific Membrane Antigen monoclonal antibody),Pyrazoloacridine (NSC-366140, PD-115934), Quinine, R115777 (Zarnestra®),Raloxifene hydrochloride (Evista®, Keoxifene hydrochloride), Raltitrexed(Tomudex®, ZD-1694), Rebeccamycin, Retinoic acid, R-flurbiprofen(Flurizan®, E-7869, MPC-7869), RFS-2000 (9-nitrocamptothecan, 9-NC,Rubitecan®), Rituximab® (Rituxan®, anti-CD₂₀ MAb), RSR-13 (GSJ-61),Satraplatin (BMS-182751, JM-216), SCH 6636, SCH-66336, Sizofilan® (SPG,Sizofuran®, Schizophyllan®, Sonifilan®), SKI-2053R(NSC-D644591),Sobuzoxane (MST-16, Perazolin®), Squalamine (MSI-1256F), SR-49059(vasopressin receptor inhibitor, Vla), Streptozocin (Zanosar®), SU5416(Semaxanib®, VEGF inhibitor), SU6668 (PDGF-TK inhibitor), T-67(T-138067, T-607), Talc (Sclerosol®), Tamoxifen (Nolvadex®), Taurolidine(Taurolin®), Temozolamide (Temodar®, NSC 362856), Teniposide (VM-26,Vumon®), TER-286, Testosterone (Andro®, Androderm®, Testoderm TTS®,Testoderm®, Depo-Testosterone®, Androgel®, depoAndro®), Tf-CRM107(Transferrin-CRM-107), Thalidomide and thalidomide analogs, includingbut not limited to, lenalidomide (CC-5013, REVLIMID®) and CC-4047(ACTIMID™), Theratope, Thioguanine (6-thioguanine, 6-TG), Thiotepa(triethylenethiophosphaoramide, Thioplex®), Thymosin alpha I (Zadaxin®,Thymalfasin®), Tiazofurin (Thiazole®), Tirapazamine (SR-259075, SR-4233,Tirazone®, Win-59075), TNP-470 (AGM-1470, Fumagillin), Tocladesine(8-Cl-cAMP), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®),Toremifene (Estrimex®, Fareston®), Tositumomab® (Bexxar®), Tretinoin(Retin-A®, Atragen®, ATRA, Vesanoid®), TriAb® (anti-idiotype antibodyimmune stimulator), Trilostane (Modrefen®), Triptorelin pamoate(Treistar Depot®, Decapeptyl®), Trimetrexate (Neutrexin®), Troxacitabine(BCH-204, BCH-4556, Troxatyl®), TS-1, UCN-01 (7-hydroxystaurosporine),Valrubicin (Valstar®), Vaispodar (PSC 833), Vapreotide® (BMY-41606),Vaxid (B-cell lymphoma DNA vaccine), Vinblastine (Velban®, VLB),Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®), Vindesine(Eldisine®, Fildesin®), Vinorelbine (Navelbine®), Vitaxin® (LM-609,integrin alphavbeta3 antagonistic MAb), WF10 (macrophage regulator),WHI-P131, WT1 Vaccine, XR-5000 (DACA), XR-9576 (XR-9351,P-glycoprotein/MDR inhibitor), ZD-9331, ZD-1839 (IRESSA®), andZoledronate (Zometa®).

In one embodiment, antibodies of the present invention may beadministered in combination with a taxane. In another embodiment,antibodies of the present invention may be administered in combinationwith a taxane for the treatment of cancers and premalignant conditionsthat are resistant to individual chemotherapies. In a specificembodiment, antibodies of the present invention may be administered incombination with Docetaxel (Taxotere®). In a specific embodiment,antibodies of the present invention may be administered in combinationwith Docetaxel (Taxotere®) for the treatment of cancers and premalignantconditions that are resistant to individual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a taxane. In another embodiment,agonistic antibodies of the present invention may be administered incombination with a taxane for the treatment of cancers and premalignantconditions that are resistant to individual chemotherapies. In aspecific embodiment, agonistic antibodies of the present invention maybe administered in combination with Docetaxel (Taxotere®). In a specificembodiment, agonistic antibodies of the present invention may beadministered in combination with Docetaxel (Taxotere®) for the treatmentof cancers and premalignant conditions that are resistant to individualchemotherapies

In one embodiment, antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic. Inanother embodiment, antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic forthe treatment of cancers and premalignant conditions that are resistantto individual chemotherapies. In another specific embodiment, antibodiesof the invention may be administered in combination with Carboplatin(Paraplatin®, CBDCA). In another specific embodiment, antibodies of thepresent invention may be administered in combination with Carboplatin(Paraplatin®, CBDCA) for the treatment of cancers that are resistant toindividual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic. Inanother embodiment, agonistic antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic forthe treatment of cancers and premalignant conditions that are resistantto individual chemotherapies. In another specific embodiment, agonisticantibodies of the invention may be administered in combination withCarboplatin (Paraplatin®, CBDCA). In another specific embodiment,agonistic antibodies of the present invention may be administered incombination with Carboplatin (Paraplatin®, CBDCA) for the treatment ofcancers that are resistant to individual chemotherapies.

In one embodiment, antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor. In anotherembodiment, antibodies of the present invention may be administered incombination with a topoisomerase inhibitor for the treatment of cancersand premalignant conditions that are resistant to individualchemotherapies. In a specific embodiment, antibodies of the presentinvention may be administered in combination with Irinotecan(Camptosar®, CPT-11, Topotecin®, CaptoCPT-1). In a specific embodiment,antibodies of the present invention may be administered in combinationwith Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1) for thetreatment of cancers that are resistant to individual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor. In anotherembodiment, agonistic antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor for thetreatment of cancers and premalignant conditions that are resistant toindividual chemotherapies. In a specific embodiment, agonisticantibodies of the present invention may be administered in combinationwith Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1). In aspecific embodiment, agonistic antibodies of the present invention maybe administered in combination with Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1) for the treatment of cancers and premalignantconditions that are resistant to individual chemotherapies.

In one embodiment, antibodies of the present invention may beadministered in combination with a fluoropyrimidine. In anotherembodiment, antibodies of the present invention may be administered incombination with a fluoropyrimidine for the treatment of cancers andpremalignant conditions that are resistant to individual chemotherapies.In another specific embodiment, antibodies of the invention may beadministered in combination with Fluorouracil (5-FU, Adrucil®). Inanother specific embodiment, antibodies of the present invention may beadministered in combination with Fluorouracil (5-FU, Adrucil®) for thetreatment of cancers and premalignant conditions that are resistant toindividual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a fluoropyrimidine. In anotherembodiment, agonistic antibodies of the present invention may beadministered in combination with a fluoropyrimidine for the treatment ofcancers and premalignant conditions that are resistant to individualchemotherapies. In another specific embodiment, agonistic antibodies ofthe invention may be administered in combination with Fluorouracil(5-FU, Adrucil®). In another specific embodiment, agonistic antibodiesof the present invention may be administered in combination withFluorouracil (5-FU, Adrucil®) for the treatment of cancers andpremalignant conditions that are resistant to individual chemotherapies.

In one embodiment, the antibody compositions of the invention areadministered in combination with apoptosis inducing polypeptides. In aspecific embodiment, antibodies of the invention are administered incombination with Smac (second mitochondria-derived activator ofcaspases) proteins, also known as DIABLO (direct IAP (inhibitor ofapoptosis) binding protein with low pI) (GenBank Accession No.:NP_(—)063940 which is hereby incorporated by reference in its entirety).Smac is a 239 amino acid protein. The N-terminal 55 amino acids serve asa mitochondrial targeting sequence which is cleaved after import to themitochondria. Apoptosis inducing polypeptides may be delivered usingtechniques known in the art. For example, one way to deliver Smacprotein would be through the delivery of a nucleic acid encoding eitherthe full length or mature form of Smac (amino acids 56-239 of GenBankAccession No.: NP_(—)063940, a cytosolic form that bypassesmitochondrial processing). Alternatively, antibody compositions of theinvention may be administered in combination with cell permeable,synthetic Smac peptides which are capable of inhibiting IAP proteins(e.g., those containing amino acid residues 56-62 of GenBank AccessionNo.: NP 063940; AVPIAQK as described in Chai et al., (2000) Nature406:855-862 and Fulda et al., (2002) Nature Medicine 8:808-815, both ofwhich are hereby incorporated by reference in their entireties.Alternatively, an antibody composition of the invention may beadministered in combination with a small molecule IAP inhibitorcompound. In specific embodiments, the IAP inhibitor compound mimics theactivity of SMAC by binding to one or more IAPs and blockingIAP-mediated caspase inhibition. The IAP inhibitor may be a monomer, adimer, a trimer, or a higher-order multimer. In specific embodiments thesmall molecule IAP inhibitor binds to and inhibits the activity ofX-linked inhibitor of apoptosis (XIAP). In further embodiments, anantibody composition of the invention is administered with one, two,three, or more of the IAP inhibitors described in Li et al. Science.2004305(5689):1471-4 (2004) (e.g., “Compound 3”); Schimmer et al.,Cancer Cell. 5(1):25-35 (2004) (e.g., compounds 1396-12 and 1396-34);and Zobel et al., ACS Chem. Biol. 1(8):525-33 (2006); which are herebyincorporated by reference in their entirety. Exemplary IAP inhibitorsthat may be administered in combination with an antibody compound of theinvention also include one, two, three, or more of the IAP inhibitorsdescribed in U.S. Patent Publication Nos. 20070003535; 20060264379;20060194741; 20060167066; 20060128632; 20060084611; 20060025347;20060014700; 20050261203; 20050197403; 20040077542; 20030143579;20020177557; and International Publication Nos. WO 05/094818; WO05/097791; WO 06/014361; WO 2006/069063; WO 2006/091972; WO 2006/113376;WO 05/092326; WO 05/069894; and WO 05/069888; each of which are herebyincorporated by reference in their entirety.

In one embodiment, an antibody composition of the invention isadministered in combination with a histone deacetylase inhibitor (e.g.,trichostatin A, trapoxins, depsipeptide (e.g., FK-288 and FR901228),MS-275, and the triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oicacid (CDDO) or other molecules related to CDDO, valproic acid,suberoylanilide hydroxamic acid (SAHA), pyroxamide, trapoxin,(depsipeptide), and N-acetyl dinaline (CI-994). In additionalembodiments an antibody composition of the invention is administered incombination with one, two, three, or more histone deacytelase inhibitorsselected from APHA Compound 8(3-(1-Methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide);Apicidin(Cyclo[(2S)-2-amino-8-oxodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperidinexcarbonyl]);Sodium Butyrate (Butyric Acid Sodium salt); (−)-Depudecin(4,5:8,9-Dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undeca-1,6-dienitol);Scriptaid (6-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acidhydroxyamide); Sirtinol(2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide);Trichostatin A([R-(E,E)]-7-[4-(Dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide);and histone deacetylase inhibitors described in US Patent PublicationNos. 20050131018; 20050124679; 20050222414; 20050119250; 20050171103;20070004806; 20060235012; 20050026907; 20050148613; 20060052599;20030216345; 20060235231; 20070015809; 20040224991; 20060264415;20020103192; 20060047123; 20030134865; 20060030543; 20050107384;20060058553; 20070010669; 20050176686; 20060030554; 20050165016;20060122234; 20050171347; 20050113373; 20040077698; and 20040087652; andInternational Publication No. WO 05/025619; each of which are herebyincorporated by reference in their entirety.

In one embodiment, an antibody composition of the invention isadministered in combination with a nitric oxide donor (e.g., sodiumnitroprusside, GEA 3175 (1,2,3,4-oxatriazolium,3-(3-chloro-2-methylphenyl)-5-[[(methylphenyl)sulphonyl]amino]),isosorbide dinitrate, and nitroglycerin).

In one embodiment, an antibody composition of the invention isadministered in combination with curcumin (diferuloylmethane).

In one embodiment an antibody composition of the invention isadministered in combination with resveratrol(3,5,4′-trihydroxystilbene).

In another embodiment, an antibody composition of the invention isadministered in combination with a protein kinase inhibitor. Inparticular embodiments, the protein kinase inhibitor is a multi-targetkinase inhibitor, including but not limited to Zactima™ (ZD6474),AMG706, MP-412, sorafenib, dasatinib, CEP-701 (lestaurtinib), XL647,XL999, lapatinib, MLN518 (CT53518), PKC412, ST1571, AMN107, AEE788,OSI-930, OSI-817, sunitinib (SUI 1248), and AG-013736.

In another embodiment, an antibody composition of the invention isadministered in combination with a Heat Shock Protein (HSP) Inhibitor.In a specific embodiment the HSP inhibitor is an HSP90 inhibitor, suchas geldanamycin; geldanamycin analogs (e.g.17-allylamino-17-demethoxy-geldanamycin (17AAG) and 17-aminogeldanamycin(17-AG)); and radicicol.

In another embodiment, an antibody composition of the invention isadministered in combination with a peroxisome proliferator-activatedreceptor-gamma (PPAR-gamma) antagonist, such as, but not limited to,GW9662 and T0070907.

In another embodiment, an antibody composition of the invention isadministered in combination with a AKT/mTOR signaling pathway inhibitor,such as, but not limited to, rapamycin, temsirolimus (CCI-779), AP23573,everolimus (RAD001) and MKC-1.

In another embodiment, an antibody composition of the invention isadministered in combination with a BCL-2 inhibitor, such as, but notlimited to, HA14-1, GX15-070, and ABT-263.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, prevention, amelioration and/orcure of cancers and premalignant conditions.

In further specific embodiments, antibodies of the present invention maybe administered in combination with one or more combinations oftherapeutic agents useful in the treatment, prevention, ameliorationand/or cure of cancers and premalignant conditions including, but notlimited to, 9-aminocamptothecin+G-CSF,Adriamycing+Blenoxane+Vinblastine+Dacarbazine (ABVD), BCNU(Carmustine)+Etoposide+Ara-C (Cytarabine)+Melphalen (BEAM),Bevacizumab®+Leucovorin, Bleomycin+Etoposide+Platinolg (Cisplatin)(BEP),Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+Vincristine+Procarbazine+Prednisone(BEACOPP), Bryostatin+Vincristine, Busulfan+Melphalan,Carboplatin+Cereport®, Carboplatin+Cyclophosphamide,Carboplatin+Paclitaxel, Carboplatin+Etoposide+Bleomycin (CEB),Carboplatin+Etoposide+Thiotepa, Cisplatin+Cyclophosphamide,Cisplatin+Docetaxel, Cisplatin+Doxorubicin, Cisplatin+Etoposide,Cisplatin+Gemcitabine, Cisplatin+Interferon alpha, Cisplatin+Irinotecan,Cisplatin+Paclitaxel, Cisplatin+Teniposide, Cisplatin+Vinblastine,Cisplatin+Vindesine, Cisplatin+Vinorelbine,Cisplatin+Cytarabine+Ifosfamide, Cisplatin+Ifosfamide+Vinblastine,Cisplatin+Vinblastine+Mitomycin C, Cisplatin+Vincristine+Fluorouracil,Cisplatin+Vincristine+Lomustine, Cisplatin+Vinorelbine+Gemcitabine,Cisplatin+Carmustine+Dacarbazine+Tamoxifen,Cisplatin+Cyclophosphamide+Etoposide+Vincristine, Cisplatin(Platinol®)+Oncovin®+Doxorubicin (Adriamycin®)+Etoposide (CODE),Cisplatin+Cytarabine+Ifosfamide+Etoposide+Methotrexate,Cyclophosphamide+Adriamycin® (Doxorubicin), Cyclophosphamide+Melphalan,Cyclophosphamide+SCH 6636, Cyclophosphamide+Adriamycing+Cisplatin(Platinol®) (CAP), Cyclophosphamide+Adriamycin®+Vincristine (CAV),Cyclophosphamide+Doxorubicin+Teniposide+Prednisone,Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon alpha,Cyclophosphamide+Epirubicin+Cisplatin (Platinol®) (CEP),Cyclophosphamide+Epirubicin+Fluorouracil,Cyclophosphamide+Methotrexate+Fluoruracil (CMF),Cyclophosphamide+Methotrexate+Vincristine (CMV),Cyclophosphamide+Adriamycing+Methotrexate+Fluorouracil (CAMF),Cyclophosphamide+Adriamycin®+Methotrexate+Procarbazine (CAMP),Cyclophosphamide+Adriamycin®+Vincristine+Etoposide (CAV-E),Cyclophosphamide+Adriamycing+Vincristine+Prednisone (CHOP),Cyclophosphamide+Novantrone® (Mitoxantrone)+Vincristine(Oncovorin)+Prednisone (CNOP),Cyclophosphamide+Adriamycin®+Vincristine+Prednisone+Rituximab(CHOP+Rituximab), Cyclophosphamide+Adriamycin®+Vincristine+Teniposide(CAV-T), Cyclophosphamide+Adriamycin®+Vincristine alternating withPlatinol®+Etoposide (CAV/PE), Cyclophosphamide+BCNU (Carmustine)+VP-16(Etoposide) (CBV), Cyclophosphamide+Vincristine+Prednisone (CVP),Cyclophosphamide+Oncovin®+Methotrexate+Fluorouracil (COMF),Cytarabine+Methotrexate, Cytarabine+Bleomycin+Vincristine+Methotrexate(CytaBOM), Dactinomycin+Vincristine, Dexamethasone+Cytarabine+Cisplatin(DHAP), Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE),Docetaxel+Gemcitabine, Docetaxel+Vinorelbine,Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+Prednisone(Stanford V), Epirubicin+Gemcitabine, Estramustine+Docetaxel,Estramustine+Navelbine, Estramustine+Paclitaxel,Estramustine+Vinblastine, Etoposide (Vepesid®)+Ifosfamide+Cisplatin(Platinol®) (VIP), Etoposide+Vinblastine+Adriamycin (EVA), Etoposide(Vepesid®)+Ifosfamide+Cisplatin+Epirubicin (VIC-E),Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP),Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC),Fludarabine+Mitoxantrone+Dexamethasone (FMD),Fludarabine+Dexamethasone+Cytarabine (ara-C)+Cisplatin (Platinol®)(FluDAP), Fluorouracil+Bevacizumab®, Fluorouracil+CeaVac®,Fluorouracil+Leucovorin, Fluorouracil+Levamisole,Fluorouracil+Oxaliplatin, Fluorouracil+Raltitrexed, Fluorouracil+SCH6636, Fluorouracil+Trimetrexate, Fluorouracil+Leucovorin+Bevacizumab®,Fluorouracil+Leucovorin+Oxaliplatin,Fluorouracil+Leucovorin+Trimetrexate, Fluorouracil+Oncovin®+Mitomycin C(FOMi), Hydrazine+Adriamycin®+Methotrexate (HAM), Ifosfamide+Docetaxel,Ifosfamide+Etoposide, Ifosfamide+Gemcitabine, Ifosfamide+Paclitaxel,Ifosfamide+Vinorelbine, Ifosfamide+Carboplatin+Etoposide (ICE),Ifosfamide+Cisplatin+Doxorubicin, Irinotecan+C225 (Cetuximab®),Irinotecan+Docetaxel, Irinotecan+Etoposide, Irinotecan+Fluorouracil,Irinotecan+Gemcitabine, Mechlorethamine+Oncovin®(Vincristine)+Procarbazine (MOP), Mechlorethamine+Oncovin®(Vincristine)+Procarbazine+Prednisone (MOPP),Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate+Interferonalpha, Methotrexate+Vinblastine, Methotrexate+Cisplatin, Methotrexatewith leucovorinrescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone(m-BACOD), Mitomycin C+Ifosfamide+Cisplatin (Platinol®) (MIP), MitomycinC+Vinblastine+Paraplatin® (MVP), Mitoxantrone+Hydrocortisone,Mitoxantrone+Prednisone, Oncovin®+SCH 6636, Oxaliplatin+Leucovorin,Paclitaxel+Doxorubicin, Paclitaxel+SCH 6636, Paraplatin®+Docetaxel,Paraplatin®+Etoposide, Paraplatin®+Gemcitabine, Paraplatin®+Interferonalpha, Paraplatin®+Irinotecan, Paraplatin®+Paclitaxel,Paraplatin®+Vinblastine, Carboplatin (Paraplatin®)+Vincristine,Paraplatin®+Vindesine, Paraplatin®+Vinorelbine, Pemetrexeddisodium+Gemcitabine, Platinolg (Cisplatin)+Vinblastine+Bleomycin (PVB),Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide (ProMACE),Procarbazine+Lomustine, Procarbazine+Lomustine+Vincristine,Procarbazine+Lomustine+Vincristine+Thioguanine,Procarbazine+Oncovin®+CCNU®+Cyclophosphamide (POCC),Quinine+Doxorubicin, Quinine+Mitoxantrone+Cytarabine,Thiotepa+Etoposide, Thiotepa+Busulfan+Cyclophosphamide,Thiotepa+Busulfan+Melphalan, Thiotepa+Etoposide+Carmustine,Thiotepa+Etoposide+Carboplatin, Topotecan+Paclitaxel,Trimetrexate+Leucovorin, Vinblastine+Doxorubicin+Thiotepa,Vinblastine+Bleomycin+Etoposide+Carboplatin,Vincristine+Lomustine+Prednisone, Vincristine(Oncovin®)+Adriamycin®+Dexamethasone (VAD), Vincristine(Oncovin®)+Adriamycin®+Procarbazine (VAP),Vincristine+Dactinomycin+Cyclophosphamide, and Vinorelbine+Gemcitabine.

In one embodiment, antibodies of the present invention may beadministered in combination with a taxane and a platinum-basedchemotherapeutic. In another embodiment, antibodies of the presentinvention may be administered in combination with a taxane and aplatinum-based chemotherapeutic for the treatment of cancers andpremalignant conditions that are resistant to individual chemotherapies.In a specific embodiment, antibodies of the present invention may beadministered in combination with Docetaxel (Taxotere®) and Carboplatin(Paraplatin®, CBDCA). In another specific embodiment, antibodies of thepresent invention may be administered in combination with Docetaxel(Taxotere®) and Carboplatin (Paraplatin®, CBDCA) for the treatment ofcancers and premalignant conditions that are resistant to individualchemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a taxane and a platinum-basedchemotherapeutic. In another embodiment, agonistic antibodies of thepresent invention may be administered in combination with a taxane and aplatinum-based chemotherapeutic for the treatment of cancers andpremalignant conditions that are resistant to individual chemotherapies.In a specific embodiment, agonistic antibodies of the present inventionmay be administered in combination with Docetaxel (Taxotere®) andCarboplatin (Paraplatin®, CBDCA). In another specific embodiment,agonistic antibodies of the present invention may be administered incombination with Docetaxel (Taxotere®) and Carboplatin (Paraplatin®,CBDCA) for the treatment of cancers and premalignant conditions that areresistant to individual chemotherapies.

In one embodiment, antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor and afluoropyrimidine. In another embodiment, antibodies of the presentinvention may be administered in combination with a topoisomeraseinhibitor and a fluoropyrimidine for the treatment of cancers andpremalignant conditions that are resistant to individual chemotherapies.In a specific embodiment, antibodies of the present invention may beadministered in combination with Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1) and Fluorouracil (5-FU, Adrucil®). In anotherspecific embodiment, antibodies of the present invention may beadministered in combination with Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1) and Fluorouracil (5-FU, Adrucil®) for thetreatment of cancers and premalignant conditions that are resistant toindividual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with: a topoisomerase inhibitor and afluoropyrimidine. In another embodiment, agonistic antibodies of thepresent invention may be administered in combination with atopoisomerase inhibitor and a fluoropyrimidine for the treatment ofcancers and premalignant conditions that are resistant to individualchemotherapies. In a specific embodiment, agonistic antibodies of thepresent invention may be administered in combination with Irinotecan(Camptosar®, CPT-11, Topotecin®, CaptoCPT-1) and Fluorouracil (5-FU,Adrucil®). In another specific embodiment, agonistic antibodies of thepresent invention may be administered in combination with Irinotecan(Camptosar®, CPT-11, Topotecin®, CaptoCPT-1) and Fluorouracil (5-FU,Adrucil®) for the treatment of cancers and premalignant conditions thatare resistant to individual chemotherapies.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedcombinations of therapeutic agents in the treatment, prevention,amelioration and/or cure of cancers and premalignant conditions.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents described above to treat, prevent,ameliorate and/or cure cancers and premalignant conditions of any tissueknown to express DR4 receptor. In preferred embodiments, agonisticantibodies of the present invention are administered in combination withone or more therapeutic agents described above to treat, prevent,ameliorate and/or cure cancers and premalignant conditions of any tissueknown to express DR4 receptor.

Tissues known to express DR4 receptor include, but are not limited to,heart, placenta, liver, pancreas, spleen, thymus, prostate, testis,ovary, stomach, small intestine, colon, kidney, bone marrow, skin,blood, tonsil and palate.

In specific embodiments antibodies of the present invention may beadministered in combination with one or more therapeutic agents, asdescribed above, in the treatment, prevention, amelioration and/or cureof solid tissue cancers and premalignant conditions (e.g., skin cancer,prostate cancer, pancreatic cancer, hepatic cancer, lung cancer, ovariancancer, colorectal cancer, head and neck tumors, breast tumors,endothelioma, osteoblastoma, osteoclastoma, Ewing's sarcoma, andKaposi's sarcoma), as well as hematological cancers (e.g., leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin'slymphoma, multiple myeloma).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more therapeutic agents, asdescribed above, in the treatment, prevention, amelioration and/or cureof solid tissue cancers and premalignant conditions (e.g., skin cancer,prostate cancer, pancreatic cancer, hepatic cancer, lung cancer, ovariancancer, colorectal cancer, head and neck tumors, breast tumors,endothelioma, osteoblastoma, osteoclastoma, Ewing's sarcoma, andKaposi's sarcoma), as well as hematological cancers (e.g., leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin'slymphoma, multiple myeloma).

In specific embodiments antibodies of the present invention are used totreat, ameliorate and/or prevent skin cancers and premalignantconditions including basal cell carcinoma, squamous cell carcinoma andmalignant melanoma. Antibodies of the present invention may be used incombination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent skincancers and premalignant conditions.

In preferred embodiments agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent skin cancers andpremalignant conditions including basal cell carcinoma, squamous cellcarcinoma and malignant melanoma. Agonistic antibodies of the presentinvention may be used in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent skin cancers and premalignant conditions.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of skincancers and premalignant conditions including, but not limited to,Bleomycin (Blenoxane®), Carmustine (DTI-015, BCNU, BiCNU, GliadelWafer®), Cisplatin (Platinol®, CDDP), Dacarbazine (DTIC), Interferonalpha 2b (Intron A®), Interleukin-2 (ProleiukinR®), Tamoxifen(Nolvadex®), Temozolamide (Temodar®, NSC 362856), Vinblastine (Velban®,VLB), Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®), andVindesine (Eldisine®, Fildesin®). Combinations of therapeutic agentsuseful in the treatment of skin cancers include, but are not limited to,Cisplatin+Carmustine+Dacarbazine+Tamoxifen.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofskin cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent head and neck cancersincluding brain cancers, and premalignant conditions. Antibodies of thepresent invention may be used in combination with one or more surgicaland/or radiological procedures and/or therapeutic agents to treat,ameliorate and/or prevent head and neck cancers including brain cancers,and premalignant conditions. Brain cancers which may be treated usingantibodies of the present invention include, but are not limited to,gliomas such as astrocytomas and oligodendromas, non-glial tumors suchas neuronal, meningeal, ependymal and choroid plexus cell tumors, andmetastatic brain tumors such as those originating as breast, lung,prostate and skin cancers.

In further preferred embodiments, agonistic antibodies of the presentinvention are used to treat, ameliorate and/or prevent head and neckcancers including brain cancers, and premalignant conditions. Agonisticantibodies of the present invention may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent head and neck cancersincluding brain cancers, and premalignant conditions. Brain cancerswhich may be treated using agonistic antibodies of the present inventioninclude, but are not limited to, gliomas such as astrocytomas andoligodendromas, non-glial tumors such as neuronal, meningeal, ependymaland choroid plexus cell tumors, and metastatic brain tumors such asthose originating as breast, lung, prostate and skin cancers.

In one preferred embodiment, agonistic antibodies of the invention areused to treat brain tumors. In a further preferred embodiment, agonisticantibodies of the invention are used to treat glioblastoma multiforme.

Antibodies of the present invention may be administered in combinationwith one or more radiological procedures useful in the treatment ofbrain cancers including, but not limited to, external beam radiationtherapy, stereotactic radiation therapy, conformal radiation therapy,intensity-modulated radiation therapy (IMRT), and radiosurgery.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more radiological proceduresuseful in the treatment of brain cancers including, but not limited to,external beam radiation therapy, stereotactic radiation therapy,conformal radiation therapy, intensity-modulated radiation therapy(IMRT), and radiosurgery.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of braincancers including, but not limited to, Bleomycin (Blenoxane®), Busulfan(Busulfex®, Myleran®), Carboplatin (Paraplatin®, CBDCA), Carmustine(DTI-015, BCNU, BiCNU, Gliadel Wafer®), Cisplatin (Platinol®, CDDP),Cisplatin-epinephrine gel (IntraDose®, FocaCist®), Cyclophosphamide(Cytoxan®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside,DepoCyt®), Dacarbazine (DTIC®), Dactinomycin (Cosmegen®), Daunorubicin(Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®), Docetaxel(Taxotere®, Taxane®), Dexamethasone (Decadron®), Etoposide phosphate(Etopophos®), Etoposide (VP-16, Vepesid®), Fluorouracil (5-FU,Adrucil®), Hydroxyurea (Hydrea®), Ifosfamide (IFEX®), Lomustine (CCNU®,CeeNU®), Melphalan (L-PAM, Alkeran®, Phenylalanine mustard),Mercaptopurine (6-mercaptopurine, 6-MP), Methchlorethamine (NitrogenMustard, HN₂, Mustargen®), Methotrexate® (MTX, Mexate®, Folex®),Paclitaxel (Paxene®), Taxol®), Paclitaxel-DHA (Taxoprexin®),Procarbazine (Matulane®)), Temozolamide (Temodar®, NSC 362856),Teniposide (VM-26, Vumon®), Thioguanine (6-thioguanine, 6-TG), Thiotepa(triethylenethiophosphaoramide), Topotecan (Hycamtin®, SK&F-104864,NSC-609699, Evotopin®), and Vincristine (Oncovin®, Onco TCS®, VCR,Leurocristine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbrain cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment of braincancers which may be administered in combination with antibodies of thepresent invention include, but are not limited to, 81C6 (Anti-tenascinmonoclonal antibody), BIBX-1382, Cereport® (Lobradimil®, RMP-7),Cilengitide® (EMD-121974, integrin alphavbeta3 antagonist), CMT-3(Metastat®), Cotara®) (chTNT-1/B, [¹³¹I]-chTNT-1/B), CP IL-4-toxin (IL-4fusion toxin), Fenretinideg (4HPR), Fotemustine (Muphoran®,Mustophoran®), Gemcitabine (Gemto®, Gemzar®), Hypericin® (VIMRxyn®),Imatinib mesylate (STI-571, Imatinib®, Glivec®, Gleevec®, Ab1 tyrosinekinase inhibitor), Irinotecan (Camptosar®, CPT-11, Topotecin®,CaptoCPT-1), Leflunomide (SU-101, SU-0200), Mivobulin isethionate(CI-980), O6-benzylguanine (BG, Procept®), Prinomastat® (AG-3340, MMPinhibitor), R115777 (Zamestra®), SU6668 (PDGF-TK inhibitor), T-67(T-138067, T-607), Tamoxifen (Nolvadex®), Tf-CRM107(Transferrin-CRM-107), Thalidomide and thalidomide analogs, includingbut not limited to, lenalidomide (CC-5013, REVLIMID®) and CC-4047(ACTIMID™), Tiazofurin (Thiazole®), Vapreotide® (BMY-41606), Vinorelbine(Navelbine®), and XR-5000 (DACA).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbrain cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofbrain cancers which may be administered in combination with antibodiesof the present invention include, but are not limited to,Busulfan+Melphalan, Carboplatin+Cereport®, Carboplatin+Etoposide,Carboplatin+Etoposide+Thiotepa, Cisplatin+Etoposide,Cisplatin+Cytarabine+Ifosfamide, Cisplatin+Vincristine+Lomustine,Cisplatin+Cyclophosphamide+Etoposide+Vincristine,Cisplatin+Cytarabine+Ifosfamide+Etoposide+Methotrexate,Cyclophosphamide+Melphalan, Cytarabine+Methotrexate,Dactinomycin+Vincristine, Mechlorethamine+Oncovin®(Vincristine)+Procarbazine (MOP), Mechlorethamine+Oncovin®(Vincristine)+Procarbazine+Prednisone (MOPP), Carboplatin(Paraplatin®)+Etoposide, Carboplatin (Paraplatin®)+Vincristine,Procarbazine+Lomustine, Procarbazine+Lomustine+Vincristine,Procarbazine+Lomustine+Vincristine+Thioguanine, Thiotepa+Etoposide,Thiotepa+Etoposide+Carmustine, Thiotepa+Etoposide+Carboplatin,Vinblastine+Bleomycin+Etoposide+Carboplatin, andVincristine+Lomustine+Prednisone.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedcombinations of therapeutic agents in the treatment, amelioration and/orprevention of brain cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent breast cancer andpremalignant conditions. Antibodies of the present invention may be usedin combination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent breastcancer and premalignant conditions. Breast cancers which may be treatedusing antibodies of the present invention include, but are not limitedto, ductal carcinoma, stage I, stage II, stage III and stage IV breastcancers as well as invasive breast cancer and metastatic breast cancer.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent breast cancer andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventbreast cancer and premalignant conditions. Breast cancers which may betreated using agonistic antibodies of the present invention include, butare not limited to, ductal carcinoma, stage I, stage II, stage III andstage IV breast cancers as well as invasive breast cancer and metastaticbreast cancer.

In one preferred embodiment, agonistic antibodies of the invention areused to treat metastatic breast cancer.

Antibodies of the present invention may be administered in combinationwith one or more surgical and/or radiological procedures useful in thetreatment of breast cancer and premalignant conditions.

In preferred embodiments, agonistic antibodies of the present inventionmay be administered in combination with one or more surgical and/orradiological procedures useful in the treatment of breast cancer andpremalignant conditions.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of breastcancer and premalignant conditions including, but not limited to,Amifostine (Ethyol®), Aminoglutethimide (Cytadren®), Anastrozole(Arimidex®), Bleomycin (Blenoxane®), Capecitabine (Xeloda®,Doxifluridine®, oral 5-FU), Cisplatin (Platinol®, CDDP),Cisplatin-epinephrine gel (IntraDose®, FocaCist®), Cyclophosphamide(Cytoxan®, Neosar®, CTX), Docetaxel (Taxotere®, Taxane®), Doxorubicin(Adriamycin®, Doxil®, Rubex®), Epirubicin (Ellence®, EPI, 4′epi-doxorubicin), Exemestane (Aromasin®, Nikidess®), Fadrozole (Afema®,Fadrozole hydrochloride, Arensin®), Fluorouracil (5-FU, Adrucil®,Fluoroplex®, Efudex®), Herceptin® (Trastuzumab®, Anti-HER-2 monoclonalantibody, Anti-EGFR-2 MAb), Ifosfamide (IFEX®), Letrozole (Femara®),Leucovorin (Leucovorin®, Wellcovorin®), Mechlorethamine (NitrogenMustard, HN₂, Mustargen®), Megestrol acetate (Megace®, Pallace®),Melphalan (L-PAM, Alkeran®, Phenylalanine mustard), Methotrexate® (MTX,Mexate®, Folex®), Methyltestosterone (Android-108, Testred®, Virilon®),Mitomycin C (Mitomycin®, Mutamycin®, Mito Extra®), Orzel®(Tegafur+Uracil+Leucovorin), Paclitaxel (Paxene®, Taxol®), Sobuzoxane(MST-16, Perazolin®), Tamoxifen (Nolvadex®), Testosterone (Andro®,Androderm®, Testoderm TTS®, Testoderm®, Depo-Testosterone®, Androgel®,depoAndro®), Vinblastine (Velban®, VLB), Vincristine (Oncovin®, OncoTCS®, VCR, Leurocristine®), and Vinorelbine (Navelbine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbreast cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment of breastcancer and premalignant conditions which may be administered incombination with antibodies of the present invention include, but arenot limited to, Aldesleukin (IL-2, Proleukin®), Altretamine (Hexylen®,hexamethylmelamine, Hexastat®), Angiostatin, Annamycin (AR-522,annamycin LF, Aronex®), Biricodar dicitrate (Incel®, Incel MDRInhibitor), Boronated Protoporphyrin Compound (PDIT, PhotodynanicImmunotherapy), Bryostatin-1 (Bryostatin, BMY-45618, NSC-339555),Busulfan (Busulfex®, Myleran®), Carmustine (DTI-015, BCNU, BiCNU,Gliadel Wafer®), D-limonene, Dacarbazine (DTIC), Daunorubicin(Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®), Dolastatin-10(DOLA-10, NSC-376128), DPPE, DX-8951f (DX-8951), EMD-121974, Endostatin,EO9 (EO1, EO4, EO68, EO70, EO72), Etoposide phosphate (Etopophos®),Etoposide (VP-16, Vepesid®V), Fluasterone, Fludarabine (Fludara®, FAMP),Flutamide (Eulexin®), Formestane (Lentaron®), Fulvestrant (Faslodex®),Galarubicin hydrochloride (DA-125), Gemcitabine (Gemto®, Gemzar®),Her-2/Neu vaccine, Hydroxyurea (Hydrea®), Idarubicin (Idamycin®, DMDR,IDA), Interferon alpha 2a (Intron A®), Interferon gamma(Gamma-interferon, Gamma 100®, Gamma-IF), Irinotecan (Camptosar®,CPT-11, Topotecin®, CaptoCPT-1), Ketoconazole (Nizoral®), KRN-8602 (MX,MY-5, NSC-619003, MX-2), L-asparaginase (Elspar®), Leuprolide acetate(Viadur®, Lupron®), Lomustine (CCNU®, CeeNU®), LY-335979, Mannan-MUC1vaccine, 2-Methoxyestradiol (2-ME, 2-ME2), Mitoxantrone (Novantrone®,DHAD), Motexafin Lutetium (Lutrin®, Optrin®, Lu-Tex®, lutetiumtexaphyrin, Lucyn®, Antrin®), MPV-2213ad (Finrozole®), MS-209, Muc-1vaccine, NaPro Paclitaxel, Perillyl alcohol (perilla alcohol, perillicalcohol, perillol, NSC-641066), Pirarubicin (THP), Procarbazine(Matulane®), Providence Portland Medical Center Breast Cancer Vaccine,Pyrazoloacridine (NSC-366140, PD-115934), Raloxifene hydrochloride(Evista®, Keoxifene hydrochloride), Raltitrexed (Tomudex®, ZD-1694),Rebeccamycin, Streptozocin (Zanosar®), Temozolamide (Temodar®, NSC362856), Theratope, Thiotepa (triethylenethiophosphaoramide, Thioplex®),Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®), Toremifene(Estrimex®, Fareston®), Trilostane (Modrefen®), and XR-9576 (XR-9351,P-glycoprotein/MDR inhibitor).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbreast cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofbreast cancer which may be administered in combination with antibodiesof the present invention include, but are not limited to,Cyclophosphamide+Adriamycin® (Doxorubicin),Cyclophosphamide+Epirubicin+Fluorouracil,Cyclophosphamide+Methotrexate+Fluorouracil (CMF),Paclitaxel+Doxorubicin, and Vinblastine+Doxorubicin+Thiotepa.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of breast cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent lung cancer andpremalignant conditions. Antibodies of the present invention may be usedin combination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent lungcancer and premalignant conditions. Lung cancer which may be treatedusing antibodies of the present invention includes, but is not limitedto, non-small cell lung cancer (NSCLC) including early stage NSCLC(i.e., Stage IA/IB and Stage IIA/IIB), Stage IIIA NSCLC, Stage IIA(unresectable)/IIIB NSCLC and Stage IV NSCLC, small cell lung cancer(SCLC) including limited stage SCLC and extensive stage SCLC as well asMalignant Pleural Mesothelioma.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent lung cancer andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventlung cancer and premalignant conditions. Lung cancer which may betreated using agonistic antibodies of the present invention includes,but is not limited to, non-small cell lung cancer (NSCLC) includingearly stage NSCLC (i.e., Stage IA/IB and Stage IIA/IIB), Stage IIIANSCLC, Stage IIA (unresectable)/IIIB NSCLC and Stage IV NSCLC, smallcell lung cancer (SCLC) including limited stage SCLC and extensive stageSCLC as well as Malignant Pleural Mesothelioma.

In one preferred embodiment, agonistic antibodies of the invention areused to treat non-small cell lung cancers.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of lungcancer and premalignant conditions including, but not limited to, BAY43-9006 (Raf kinase inhibitor), Carboplatin (Paraplatin®, CBDCA),Chlorambucil (Leukeran®), Cisplatin (Platinol®, CDDP),Cisplatin-epinephrine gel (IntraDose®, FocaCist®), Cyclophosphamide(Cytoxan®, Neosar®, CTX), Docetaxel (Taxotere®, Taxane®), Doxorubicin(Adriamycin®, Doxil®, Rubex®), Edatrexate, Epirubicin (Ellence®, EPI, 4′epi-doxorubicin), Etoposide phosphate (Etopophos®), Etoposide (VP-16,Vepesid®), Gemcitabine (Gemto®, Gemzar®), Herceptin® (Trastuzumab®,Anti-HER-2 monoclonal antibody, Anti-EGFR-2 MAb), Ifosfamide (IFEX®),Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1), Lomustine(CCNU®, CeeNU®), Mechlorethamine (Nitrogen Mustard, HN₂, Mustargen®),Melphalan (L-PAM, Alkeran®, Phenylalanine mustard), Methotrexate® (MTX,Mexate®, Folex®), Mitomycin C (Mitomycin®, Mutarnycin®, Mito Extra®),Paclitaxel (Paxene®, Taxol®), Paclitaxel-DHA (Taxoprexin®), Porfimersodium (Photofrin®), Procarbazine (Matulane®), SKI-2053R(NSC-D644591),Teniposide (VM-26, Vumon®), Topotecan (Hycamtin®, SK&F-104864,NSC-609699, Evotopin®V), Vinblastine (Velban®, VLB), Vincristine(Oncovin®, Onco TCS®, VCR, Leurocristine®), Vindesine (Eldisine®,Fildesin®), and Vinorelbine (Navelbine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention oflung cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment of lungcancer and premalignant conditions which may be administered incombination with antibodies of the present invention include, but arenot limited to, ABX-EGF (anti-EGFr MAb), Acetyldinaline (CI-994),AG-2034 (AG-2024, AG-2032, GARFT [glycinamide ribonucleosidetransformylase] inhibitor), Alanosine, Aminocamptothecin (9-AC,9-Aminocamptothecin, NSC 603071), Angiostatin, Aplidine (Aplidin®,Aplidina®), BBR 3464, Bexarotene (Targretin®, LGD1069), BIBH-1 (Anti-FAPMAb), BIBX-1382, BLP-25 (MUC-1 peptide), Bryostatin-1 (Bryostatin®,BMY-45618, NSC-339555), Budesonide (Rhinocort®), C225 (IMC-225, EGFRinhibitor, Anti-EGFr MAb, Cetuximab®), Capecitabine (Xeloda®,Doxifluridine®, oral 5-FU), Carboxyamidotriazole (NSC 609974, CAI,L-651582), CEA-cide® (Labetuzumab®, Anti-CEA monoclonal antibody,hMN-14), Cereport® (Lobradimil®, RMP-7), CI-1033 (Pan-erbB RTKinhibitor), Cilengitide® (EMD-121974, integrin alphavbeta3 antagonist),9-cis retinoic acid (9-cRA), Cisplatin-liposomal (SPI-077), CMB-401(Anti-PEM MAb/calicheamycin), CMT-3 (Metastat®), CP-358774 (Tarceva®,OS1-774, EGFR inhibitor), CT-2584 (Apra®), DAB389-EGF (EGF fusiontoxin), DeaVac® (CEA anti-idiotype vaccine), Decitabine(5-aza-2′-deoxyytidine), Diethylnorspermine (DENSPM),Dihydro-5-azacytidine, EGF-P64k Vaccine, Endostatin, Etanidazole(Radinyl®), Exetecan mesylate (DX-8951, DX-8951f), Exisulind (SAAND,Aptosyn®, cGMP-PDE2 and 5 inhibitor), FK-317 (FR-157471, FR-70496),Flavopiridol (HMR-1275), Fotemustine (Muphoran®, Mustophoran®), G3139(Genasense®, GentaAnticode®, Bcl-2 antisense), Gadolinium texaphyrin(Motexafin gadolinium, Gd-Tex®, Xcytrin®), GBC-590, GL331, Galarubicinhydrochloride (DA-125), Glufosfamide®S(O-D-glucosyl-isofosfamidemustard, D19575, INN), GVAX (GM-CSF gene therapy), INGN-101 (p53 genetherapy/retrovirus), INGN-201 (p53 gene therapy/adenovirus), Irofulven(MGI-114), ISIS-2053, ISIS-3521 (PKC-alpha antisense), ISIS-5132(K-ras/raf antisense), Isotretinoin (13-CRA, 13-cis retinoic acid,Accutane®), Lometrexol (T-64, T-904064), Marimastat® (BB-2516, TA-2516,MMP inhibitor), MDX-447 (BAB-447, EMD-82633, H-447,anti-EGFr/FcGammaR1r), MGV, Mitumomab® (BEC-2, EMD-60205), Mivobulinisethionate (CI-980), Neovastat® (AE-941, MMP inhibitor), NPI-0052(proteasome inhibitor), Onconase (Ranpimase®), Onyx-015 (p53 genetherapy), Pemetrexed disodium (Alimta®, MTA, multitargeted antifolate,LY 231514), Pivaloyloxymethyl butyrate (AN-9, Pivanex®), Prinomastat®)(AG-3340, MMP inhibitor), PS-341 (LDP-341, 26S proteasome inhibitor),Pyrazoloacridine (NSC-366140, PD-115934), R115777 (Zarnestra®),Raltitrexed (Tomudex®, ZD-1694), R-flurbiprofen (Flurizan®, E-7869,MPC-7869), RFS-2000 (9-nitrocamptothecan, 9-NC, Rubitecan®), RSR-13(GSJ-61), Satraplatin (BMS-182751, JM-216), SCH-66336, Sizofilan® (SPG,Sizofuran®, Schizophyllan®, Sonifilan®), Squalamine (MSI-1256F),SR-49059 (vasopressin receptor inhibitor, V1a), SU5416 (Semaxanib®, VEGFinhibitor), Taurolidine (Taurolin®), Temozolamide (Temodar®, NSC362856), Thalidomide and thalidomide analogs, including but not limitedto, lenalidomide (CC-5013, REVLIMID®) and CC-4047 (ACTIMID™) Thymosinalpha I (Zadaxin®, Thymalfasin®), Tirapazamine (SR-259075, SR-4233,Tirazone®, Win-59075), TNP-470 (AGM-1470), TriAb® (anti-idiotypeantibody immune stimulator), Tretinoin (Retin-A®, Atragen®, ATRA,Vesanoid®), Troxacitabine (BCH-204, BCH-4556, Troxatyl®), Vitaxin®(LM-609, integrin alphavbeta3 antagonistic MAb), XR-9576(P-glycoprotein/MDR inhibitor), and ZD-1839 (IRESSA®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention oflung cancers and premalignant conditions.

In one embodiment, antibodies of the present invention may beadministered in combination with a taxane. In another embodiment,antibodies of the present invention may be administered in combinationwith a taxane for the treatment of lung cancers, such as non-small celllung cancer, that are resistant to individual chemotherapies. In aspecific embodiment, antibodies of the present invention may beadministered in combination with Docetaxel (Taxotere®). In a specificembodiment, antibodies of the present invention may be administered incombination with Docetaxel (Taxotere®) for the treatment of lungcancers, such as non-small cell lung cancer, that are resistant toindividual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a taxane. In another embodiment,agonistic antibodies of the present invention may be administered incombination with a taxane for the treatment of lung cancers, such asnon-small cell lung cancer, that are resistant to individualchemotherapies. In a specific embodiment, agonistic antibodies of thepresent invention may be administered in combination with Docetaxel(Taxotere®). In a specific embodiment, agonistic antibodies of thepresent invention may be administered in combination with Docetaxel(Taxotere®) for the treatment of lung cancers, such as non-small celllung cancer, that are resistant to individual chemotherapies.

In one embodiment, antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic. Inanother embodiment, antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic forthe treatment of lung cancers, such as non-small cell lung cancer, thatare resistant to individual chemotherapies. In another specificembodiment, antibodies of the invention may be administered incombination with Carboplatin (Paraplatin®, CBDCA). In another specificembodiment, antibodies of the present invention may be administered incombination with Carboplatin (Paraplatin®, CBDCA) for the treatment oflung cancers, such as non-small cell lung cancer, that are resistant toindividual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic. Inanother embodiment, agonistic antibodies of the present invention may beadministered in combination with a platinum-based chemotherapeutic forthe treatment of lung cancers, such as non-small cell lung cancer, thatare resistant to individual chemotherapies. In another specificembodiment, agonistic antibodies of the invention may be administered incombination with Carboplatin (Paraplatin®, CBDCA). In another specificembodiment, agonistic antibodies of the present invention may beadministered in combination with Carboplatin (Paraplatin®, CBDCA) forthe treatment of lung cancers, such as non-small cell lung cancer, thatare resistant to individual chemotherapies.

Preferred combinations of therapeutic agents useful in the treatment oflung cancer and premalignant conditions which may be administered incombination with antibodies of the present invention include, but arenot limited to, Cisplatin+Docetaxel, Cisplatin+Etoposide,Cisplatin+Gemcitabine, Cisplatin+Interferon alpha, Cisplatin+Irinotecan,Cisplatin+Paclitaxel, Cisplatin+Teniposide, Cisplatin+Vinblastine,Cisplatin+Vindesine, Cisplatin+Vinorelbine,Cisplatin+Vinblastine+Mitomycin C, Cisplatin+Vinorelbine+Gemcitabine,Cisplatin (Platinol®)+Oncovin®+Doxorubicin (Adriamycin®)+Etoposide(CODE), Cyclophosphamide+Adriamycin®+Cisplatin (Platinol®) (CAP),Cyclophosphamide+Adriamycin®+Vincristine (CAV),Cyclophosphamide+Epirubicin+Cisplatin (Platinol®) (CEP),Cyclophosphamide+Methotrexate+Vincristine (CMV),Cyclophosphamide+Adriamycin®, Methotrexate+Fluorouracil (CAMF),Cyclophosphamide+Adriamycin®, Methotrexate+Procarbazine (CAMP),Cyclophosphamide+Adriamycin®, Vincristine+Etoposide (CAV-E),Cyclophosphamide+Adriamycin®, Vincristine+Teniposide (CAV-T),Cyclophosphamide+Oncovin®, Methotrexate+Fluorouracil (COMF),Cyclophosphamide+Adriamycin®+Vincristine, alternating withCisplatin+Etoposide (CAV/PE), Docetaxel+Gemcitabine,Docetaxel+Vinorelbine, Etoposide (Vepesid®)+Ifosfamide+Cisplatin(Platinol®) (VIP), Etoposide (Vepesid®)+Ifosfamide, Cisplatin+Epirubicin(VIC-E), Fluorouracil+Oncovin®+Mitomycin C (FOMi),Hydrazine+Adriamycin®+Methotrexate (HAM), Ifosfamide+Docetaxel,Ifosfamide+Etoposide, Ifosfamide+Gemcitabine, Ifosfamide+Paclitaxel,Ifosfamide+Vinorelbine, Ifosfamide+Carboplatin+Etoposide (ICE),Irinotecan+Docetaxel, Irinotecan+Etoposide, Irinotecan+Gemcitabine,Methotrexate+Cisplatin, Methotrexate+Interferon alpha,Methotrexate+Vinblastine, Mitomycin C+Ifosfamide+Cisplatin (Platinol®)(MIP), Mitomycin C+Vinblastine+Paraplatin® (MVP), Paraplatin®+Docetaxel,Paraplatin®+Etoposide, Paraplatin®+Gemcitabine, Paraplatin®+Interferonalpha, Paraplatin®+Irinotecan, Paraplatin®+Paclitaxel,Paraplatin®+Vinblastine, Paraplatin®+Vindesine, Paraplatin®+Vinorelbine,Procarbazine+Oncovin®+CCNU® (Lomustine)+Cyclophosphamide (POCC),Vincristine (Oncovin®)+Adriamycin®+Procarbazine (VAP), andVinorelbine+Gemcitabine.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of lung cancers and premalignant conditions.

In one embodiment, antibodies of the present invention may beadministered in combination with a taxane and a platinum-basedchemotherapeutic. In another embodiment, antibodies of the presentinvention may be administered in combination with a taxane and aplatinum-based chemotherapeutic for the treatment of lung cancers, suchas non-small cell lung cancer, that are resistant to individualchemotherapies. In a specific embodiment, antibodies of the presentinvention may be administered in combination with Docetaxel (Taxotere®)and Carboplatin (Paraplatin®, CBDCA). In another specific embodiment,antibodies of the present invention may be administered in combinationwith Docetaxel (Taxotere®) and tin (Paraplatin®, CBDCA) for thetreatment of lung cancers, such as non-small cell lung cancer, that areresistant to chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a taxane and a platinum-basedchemotherapeutic. In another embodiment, agonistic antibodies of thepresent invention may be administered in combination with a taxane and aplatinum-based chemotherapeutic for the treatment of lung cancers, suchas non-small cell lung cancer, that are resistant to individualchemotherapies. In a specific embodiment, agonistic antibodies of thepresent invention may be administered in combination with Docetaxel(Taxotere®) and Carboplatin (Paraplatin®, CBDCA). In another specificembodiment, agonistic antibodies of the present invention may beadministered in combination with Docetaxel (Texatere®) and Carboplatin(Paraplatin®, CBDCA) for the treatment of lung cancers, such asnon-small cell lung cancer, that are resistant to individualchemotherapies.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent colorectal cancer andpremalignant conditions. Antibodies of the present invention may be usedin combination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent colorectalcancer and premalignant conditions. Colorectal cancers which may betreated using antibodies of the present invention include, but are notlimited to, colon cancer (e.g., early stage colon cancer (stage I andII), lymph node positive colon cancer (stage 111), metastatic coloncancer (stage IV)) and rectal cancer.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent colorectal cancer andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventcolorectal cancer and premalignant conditions. Colorectal cancers whichmay be treated using agonistic antibodies of the present inventioninclude, but are not limited to, colon cancer (e.g., early stage coloncancer (stage I and II), lymph node positive colon cancer (stage III),metastic colon cancer (stage 1V)) and rectal cancer.

In one preferred embodiment, agonistic antibodies of the invention areused to treat colon cancer.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment ofcolorectal cancer and premalignant conditions including, but not limitedto, Capecitabine (Xeloda®, Doxifluridine®, oral 5-FU), Fluorouracil(5-FU, Adrucil®, Fluoroplex®, Efudex®), Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1), Leucovorin (Leucovorin®, Wellcovorin®), andLevamisole (Ergamisol®).

In one embodiment, antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor. In anotherembodiment, antibodies of the present invention may be administered incombination with a topoisomerase inhibitor for the treatment of coloncancer that is resistant to individual chemotherapies. In a specificembodiment, antibodies of the present invention may be administered incombination with Irinotecan (Camptosar®, CPT-11, Topotecin®,CaptoCPT-1). In a specific embodiment, antibodies of the presentinvention may be administered in combination with Irinotecan(Camptosar®, CPT-11, Topotecin®, CaptoCPT-1) for the treatment of coloncancer that is resistant to individual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor. In anotherembodiment, agonistic antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor for thetreatment of colon cancer that is resistant to individualchemotherapies. In a specific embodiment, agonistic antibodies of thepresent invention may be administered in combination with Irinotecan(Camptosar®, CPT-11, Topotecin®, CaptoCPT-1). In a specific embodiment,agonistic antibodies of the present invention may be administered incombination with Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1)for the treatment of colon cancer that is resistant to individualchemotherapies.

In one embodiment, antibodies of the present invention may beadministered in combination with a fluoropyrimidine. In anotherembodiment, antibodies of the present invention may be administered incombination with a fluoropyrimidine for the treatment of colon cancerthat is resistant to individual chemotherapies. In another specificembodiment, antibodies of the invention may be administered incombination with Fluorouracil (5-FU, Adrucil®). In another specificembodiment, antibodies of the present invention may be administered incombination with Fluorouracil (5-FU, Adrucil®) for the treatment ofcolon cancer that is resistant to individual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a fluoropyrimidine. In anotherembodiment, agonistic antibodies of the present invention may beadministered in combination with a fluoropyrimidine for the treatment ofcolon cancer that is resistant to individual chemotherapies. In anotherspecific embodiment, agonistic antibodies of the invention may beadministered in combination with Fluorouracil (5-FU, Adrucil®). Inanother specific embodiment, agonistic antibodies of the presentinvention may be administered in combination with Fluorouracil (5-FU,Adrucil®) for the treatment of colon cancer that is resistant toindividual chemotherapies.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofcolorectal cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofcolorectal cancer and premalignant conditions which may be administeredin combination with antibodies of the present invention include, but arenot limited to, Fluorouracil+Leucovorin, and Fluorouracil+Levamisole.

In one embodiment, antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor and afluoropyrimidine. In another embodiment, antibodies of the presentinvention may be administered in combination with a topoisomeraseinhibitor and a fluoropyrimidine for the treatment of colon cancer, thatare resistant to individual chemotherapies. In a specific embodiment,antibodies of the present invention may be administered in combinationwith Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1) andFluorouracil (5-FU, Adrucil®). In another specific embodiment,antibodies of the present invention may be administered in combinationwith Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1) andFluorouracil (5-FU, Adrucil®) for the treatment of colon cancer that isresistant to individual chemotherapies.

In one embodiment, agonistic antibodies of the present invention may beadministered in combination with a topoisomerase inhibitor and afluoropyrimidine. In another embodiment, agonistic antibodies of thepresent invention may be administered in combination with atopoisomerase inhibitor and a fluoropyrimidine for the treatment ofcolon cancer, that are resistant to individual chemotherapies. In aspecific embodiment, agonistic antibodies of the present invention maybe administered in combination with Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1) and Fluorouracil (5-FU, Adrucil®). In anotherspecific embodiment, agonistic antibodies of the present invention maybe administered in combination with Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1) and Fluorouracil (5-FU, Adrucil®) for thetreatment of colon cancer, that are resistant to individualchemotherapies.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of colorectal cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment ofcolorectal cancer which may be administered in combination withantibodies of the present invention include, but are not limited to,Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aplidine(Aplidin®, Aplidina®), Bevacizumab® (Anti-VEGF monoclonal antibody,rhuMAb-VEGF), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb, Cetuximab®),C242-DM1 (huC242-DM1), CC49-zeta gene therapy, CEA-cide® (Labetuzumab®,Anti-CEA monoclonal antibody, hMN-14), CeaVac® (MAb 3H1), CP-609754,CTP-37 (Avicine®, hCG blocking vaccine), Declopramide (Oxi-104),Eniluracil (776c85), F19 (Anti-FAP monoclonal antibody, iodinatedanti-FAP MAb), FMdC (KW-2331, MDL-101731), FUDR (Floxuridine®),Gemcitabine (Gemto®, Gemzar®), Herceptin® (Trastuzumab®, Anti-HER-2monoclonal antibody, Anti-EGFR-2 MAb), Intoplicine (RP 60475), L-778123(Ras inhibitors), Leuvectin® (cytofectin+IL-2 gene, IL-2 gene therapy),MN-14 (Anti-CEA immunoradiotherapy, 131I-MN-14, ¹⁸⁸Re-MN-14),OncoVAX-CL, OncoVAX-CL-Jenner (GA-733-2 vaccine) Orzel®(Tegafur+Uracil+Leucovorin), Oxaliplatin (Eloxatine®, Eloxatin®),Paclitaxel-DHA (Taxoprexin®), Pemetrexed disodium (Alimta®, MTA,multitargeted antifolate, LY 231514), R115777 (Zamestra®), Raltitrexed(Tomudex®, ZD-1694), SCH 66336, SU5416 (Semaxanib®, VEGF inhibitor),Tocladesine (8-Cl-cAMP), Trimetrexate (Neutrexin®), TS-1, and ZD-9331.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofcolorectal cancers and premalignant conditions.

Further exemplary combinations of therapeutic agents useful in thetreatment of colorectal cancer and premalignant conditions which may beadministered in combination with antibodies of the present inventioninclude, but are not limited to, Aminocamptothecin+G-CSF,Bevacizumab®+Fluorouracil, Bevacizumab®+Leucovorin,Bevacizumab®+Fluorouracil+Leucovorin, Cyclophosphamide+SCH 6636,Fluorouracil+CeaVac®, Fluorouracil+Oxaliplatin,Fluorouracil+Raltitrexed, Fluorouracil+SCH 6636,Fluorouracil+Trimetrexate, Fluorouracil+Leucovorin+Oxaliplatin,Fluorouracil+Leucovorin+Trimetrexate, Irinotecan+C225 (Cetuximab®),Oncovin®+SCH 6636, Oxaliplatin+Leucovorin, Paclitaxel+SCH 6636,Pemetrexed disodium+Gemcitabine, and Trimetrexate+Leucovorin.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of colorectal cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent prostrate cancer andpremalignant conditions. Antibodies of the present invention may be usedin combination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent prostatecancer. Prostate cancer which may be treated using antibodies of thepresent invention includes, but is not limited to, benign prostatichyperplasia, malignant prostate cancer (e.g., stage I, stage II, stageIII or stage IV) and metastatic prostate cancer.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent prostate cancer andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventprostate cancer and premalignant conditions. Prostate cancer which maybe treated using agonistic antibodies of the present invention includes,but is not limited to, benign prostatic hyperplasia, malignant prostatecancer (e.g., stage I, stage II, stage III or stage IV) and metastaticprostate cancer.

In one preferred embodiment, agonistic antibodies of the invention areused to treat malignant prostate cancer. In a further preferredembodiment, agonistic antibodies of the invention are used to treatmetastatic prostate cancer.

Antibodies of the present invention may be administered in combinationwith one or more surgical, radiological and/or hormonal proceduresuseful in the treatment of prostate cancer and premalignant conditionsincluding, but not limited to, prostatectomy (e.g., radical retropubicprostatectomy), external beam radiation therapy, brachytherapy,orchiectomy and hormone treatment (e.g., LHRH agonists, androgenreceptor inhibitors).

In preferred embodiments, agonistic antibodies of the present inventionmay be administered in combination with one or more surgical,radiological and/or hormonal procedures useful in the treatment ofprostate cancer including, but not limited to, prostatectomy (e.g.,radical retropubic prostatectomy), external beam radiation therapy,brachytherapy, orchiectomy and hormone treatment (e.g., LHRH agonists,androgen receptor inhibitors).

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of prostatecancer including, but not limited to, Aminoglutethimide (Cytadren®),Biclutamide (Casodex®), Cyclophosphamide (Cytoxan®, Neosar®, CTX),Diethylstilbestrol (DES), Doxorubicin (Adriamycin®, Doxil®, Rubex®),Flutamide (Eulexin®), Hydrocortisone, Ketoconazole (Nizoral®),Leuprolide acetate (Viadur®, Lupron®, Leuprogel®, Eligard®),Mitoxantrone (Novantrone®, DHAD), Nilutamide (Nilandron®), Paclitaxel(Paxene®, Taxol®), Paclitaxel-DHA (Taxoprexin®), PC SPES, Prednisone,Triptorelin pamoate (Trelstar Depot®, Decapeptyl®), and Vinblastine(Velban®, VLB).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofprostate cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment ofprostate cancer which may be administered in combination with antibodiesof the present invention include, but are not limited to, Abarelix®(Abarelix-Depot-M®, PPI-149, R-3827); Abiraterone Acetate® (CB-7598,CB-7630), ABT-627 (ET-1 inhibitor), APC-8015 (Provenge®, Dendritic celltherapy), Avorelin® (Meterelin®, MF-6001, EP-23904), CEP-701 (KT-5555),CN-706, CT-2584 (Apra®, CT-2583, CT-2586, CT-3536), GBC-590, Globo Hhexasaccharide (Globo H-KLH®), Interferon alpha 2a (Intron A®),Liarozole (Liazal, Liazol, R-75251, R-85246, Ro-85264), MDX-447(MDX-220, BAB-447, EMD-82633, H-447, anti-EGFr/FcGammaR1r), NPI-0052(proteasome inhibitor), OncoVAX-P (OncoVAX-PrPSA), PROSTVAC, PS-341(LDP-341, 26S proteasome inhibitor), PSMA MAb (Prostate SpecificMembrane Antigen monoclonal antibody), and R-flurbiprofen (Flurizan®,E-7869, MPC-7869).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofprostate cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofprostate cancer which may be administered in combination with antibodiesof the present invention include, but are not limited to,Docetaxel+Estramustine, Mitoxantrone+Hydrocortisone,Mitoxantrone+Prednisone, Navelbine+Estramustine,Paclitaxel+Estramustine, and Vinblastine+Estramustine.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of prostate cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent pancreatic cancer andpremalignant conditions. Antibodies of the present invention may be usedin combination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent pancreaticcancer and premalignant conditions. Pancreatic cancers which may betreated using antibodies of the present invention include, but are notlimited to, adenocarcinoma, endocrine (islet cell) tumors, tumorsconfined to the pancreas, locally advanced pancreatic cancer andmetastatic pancreatic cancer.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent pancreatic cancer andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventpancreatic cancer and premalignant conditions. Pancreatic cancers whichmay be treated using agonistic antibodies of the present inventioninclude, but are not limited to, adenocarcinoma, endocrine (islet cell)tumors, tumors confined to the pancreas, locally advanced pancreaticcancer and metastatic pancreatic cancer.

In one preferred embodiment, agonistic antibodies of the invention areused to treat locally advanced pancreatic cancer. In a further preferredembodiment, agonistic antibodies of the invention are used to treatmetastatic pancreatic cancer.

Antibodies of the present invention may be administered in combinationwith one or more surgical and/or radiological procedures useful in thetreatment of pancreatic cancer including, but not limited to,pancreaticoduodenumectomy (Whipple resection).

In preferred embodiments, agonistic antibodies of the present inventionmay be administered in combination with one or more surgical and/orradiological procedures useful in the treatment of pancreatic cancerincluding, but not limited to, pancreaticoduodenumectomy (Whippleresection).

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment ofpancreatic cancer including, but not limited to, Capecitabine (Xeloda®,Doxifluridine®, oral 5-FU), Cisplatin (Platinol®, CDDP), Fluorouracil(5-FU, Adrucil®, Fluoroplex®, Efudex®), Gemcitabine (Gemto®, Gemzar®),and Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofpancreatic cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofpancreatic cancer which may be administered in combination withantibodies of the present invention include, but are not limited to,Cisplatin+Gemcitabine, CP-358774+Gemcitabine, Docetaxel+Gemcitabine,Irinotecan+Fluorouracil, Irinotecan+Gemcitabine, andPaclitaxel+Gemcitabine.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of pancreatic cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment ofpancreatic cancer which may be administered in combination withantibodies of the present invention include, but are not limited to,ABX-EGF (anti-EGFr MAb), Acetyldinaline (CI-994, GOE-5549, GOR-5549,PD-130636), BMS-214662 (BMS-192331, BMS-193269, BMS-206635), BNP-1350(BNPI-1100, Karenitecins), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb,Cetuximab®), C242-DM1 (huC242-DM1, SB-408075), Carbendazin® (FB-642),Cannustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), CMT-3 (COL-3,Metastat®), CP-358774 (Tarceva®, OSI-774, EGFR inhibitor), Docetaxel(Taxotere®, Taxane®), Exetecan mesylate (DX-8951, DX-8951f),Flavopiridol (HMR-1275), Gastrimmune® (Anti-gastrin-17 immunogen,anti-g17), GBC-590, Herceptin® (Trastuzumab®, Anti-HER-2 monoclonalantibody, Anti-EGFR-2 MAb), HSPPC-96 (HSP cancer vaccine, gp96 heatshock protein-peptide complex), Irofulven (MGI-114), ISIS-2503 (Rasantisense), Onyx-015 (p53 gene therapy), Paclitaxel (Paxene®, Taxol®),Pemetrexed disodium (Alimta®, MTA, multitargeted antifolate, LY 231514),Perillyl alcohol (perilla alcohol, perillic alcohol, perillol,NSC-641066), RFS-2000 (9-nitrocamptothecan, 9-NC, Rubitecan®), andRituximab® (Rituxan®, anti-CD20 MAb).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofpancreatic cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent hepatic cancer andpremalignant conditions. Antibodies of the present invention may be usedin combination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent hepaticcancer and premalignant conditions. Hepatic cancers which may be treatedusing antibodies of the present invention include, but are not limitedto, hepatocellular carcinoma, malignant hepatoma, cholangiocarcinoma,mixed hepatocellular cholangiocarcinoma or hepatoblastoma.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent hepatic cancer andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventhepatic cancer and premalignant conditions. Hepatic cancers which may betreated using agonistic antibodies of the present invention include, butare not limited to, hepatocellular carcinoma, malignant hepatoma,cholangiocarcinoma, mixed hepatocellular cholangiocarcinoma orhepatoblastoma.

In one preferred embodiment, agonistic antibodies of the invention areused to treat hepatoblastoma. In one further preferred embodiment,agonistic antibodies of the invention are used to treat hepatocellularcarcinoma.

Antibodies of the present invention may be administered in combinationwith one or more surgical and/or radiological procedures useful in thetreatment of hepatic cancers including, but not limited to, partialhepatectomy, liver transplant, radiofrequency ablation, laser therapy,microwave therapy, cryosurgery, percutaneous ethanol injection, hepaticarterial infusion, hepatic artery ligation, chemoembolization andexternal beam radiation therapy.

In preferred embodiments, agonistic antibodies of the present inventionmay be administered in combination with one or more surgical and/orradiological procedures useful in the treatment of hepatic cancers andpremalignant conditions including, but not limited to, partialhepatectomy, liver transplant, radiofrequency ablation, laser therapy,microwave therapy, cryosurgery, percutaneous ethanol injection, hepaticarterial infusion, hepatic artery ligation, chemoembolization andexternal beam radiation therapy.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of hepaticcancer and premalignant conditions including, but not limited to,Aldesleukin (IL-2, Proleukin®), Cisplatin (Platinol®, CDDP), Doxorubicin(Adriamycin®, Doxil®, Rubex®), Etoposide phosphate (Etopophos®),Etoposide (VP-16, Vepesid®), Fluorouracil (5-FU, Adrucil®, Fluoroplex®,Efudex®), 1-131 Lipidiol®, Ifosfamide (IFEX®), Megestrol acetate(Megace®, Pallace®), Pravastatin sodium (Pravachol®), and Vincristine(Oncovin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofhepatic cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofhepatic cancer and premalignant conditions which may be administered incombination with antibodies of the present invention include, but arenot limited to, Cisplatin+Doxorubicin, Cisplatin+Etoposide,Cisplatin+Vincristine+Fluorouracil, andIfosfamide+Cisplatin+Doxorubicin.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of hepatic cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent ovarian cancer. Antibodiesof the present invention may be used in combination with one or moresurgical and/or radiological procedures and/or therapeutic agents totreat, ameliorate and/or prevent ovarian cancer. Ovarian cancers whichmay be treated using antibodies of the present invention include, butare not limited to, epithelial carcinoma, germ cell tumors and stromaltumors.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent ovarian cancer andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventovarian cancer and premalignant conditions. Ovarian cancers which may betreated using agonistic antibodies of the present invention include, butare not limited to, epithelial carcinoma, germ cell tumors and stromaltumors.

In one preferred embodiment, agonistic antibodies of the invention areused to treat germ cell tumors and premalignant conditions. In onefurther preferred embodiment, agonistic antibodies of the invention areused to treat epithelial carcinoma.

Antibodies of the present invention may be administered in combinationwith one or more surgical and/or radiological procedures useful in thetreatment of ovarian cancer and premalignant conditions including, butnot limited to, hysterectomy, oophorectomy, hysterectomy with bilateralsalpingo-oophorectomy, omentectomy, tumor debulking, external beamradiation therapy and intraperitoneal radiation therapy.

In preferred embodiments, agonistic antibodies of the present inventionmay be administered in combination with one or more surgical and/orradiological procedures useful in the treatment of ovarian cancer andpremalignant conditions including, but not limited to, hysterectomy,oophorectomy, hysterectomy with bilateral salpingo-oophorectomy,omentectomy, tumor debulking, external beam radiation therapy andintraperitoneal radiation therapy.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of ovariancancer and premalignant conditions including, but not limited to,Altretamine (Hexylen®, hexamethylmelamine, Hexastat®), Bleomycin(Blenoxane®), Carboplatin (Paraplatin®, CBDCA), Cisplatin (Platinol®,CDDP), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Dactinomycin(Cosmegen®), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Etoposidephosphate (Etopophos®), Etoposide (VP-16, Vepesid®), Fluorouracil (5-FU,Adrucil®, Fluoroplex®, Efudex®), Gemcitabine (Gemto®, Gemzar®),Ifosfamide (IFEX®), Irinotecan (Camptosar®, CPT-11, Topotecin®,CaptoCPT-1), Leucovorin (Leucovorin®, Welicovorin®), Melphalan (L-PAM,Alkeran®, Phenylalanine mustard), Paclitaxel (Paxene®, Taxol®),Tamoxifen (Nolvadex®), Vinblastine (Velban®, VLB) and Vincristine(Oncovin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofovcarian cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofovarian cancer and premalignant conditions which may be administered incombination with antibodies of the present invention include, but arenot limited to, Bleomycin+Etoposide+Platinol® (Cisplatin) (BEP),Carboplatin+Cyclophosphamide, Carboplatin+Paclitaxel,Carboplatin+Etoposide+Bleomycin (CEB), Cisplatin+Cyclophosphamide,Cisplatin+Etoposide, Cisplatin+Paclitaxel,Cisplatin+Ifosfamide+Vinblastine, Fluorouracil+Leucovorin, Platinolg(Cisplatin)+Vinblastine+Bleomycin (PVB), andVincristine+Dactinomycin+Cyclophosphamide.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of ovarian cancers and premalignant conditions.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent Ewing's sarcoma. Antibodiesof the present invention may be used in combination with one or moresurgical and/or radiological procedures and/or therapeutic agents totreat, ameliorate and/or prevent Ewing's sarcoma. Ewing's sarcoma familytumors which may be treated using antibodies of the present inventioninclude, but are not limited to, Ewing's tumor of bone (ETB),extraosseus Ewing's (EOE), primitive neuroectodermal tumors (PNET orperipheral neuroepithelioma) and Askin's tumor.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent Ewing's sarcoma. Agonisticantibodies of the present invention may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent Ewing's sarcoma. Ewing'ssarcoma family tumors which may be treated using agonistic antibodies ofthe present invention include, but are not limited to, Ewing's tumor ofbone (ETB), extraosseus Ewing's (EOE), primitive neuroectodermal tumors(PNET or peripheral neuroepithelioma) and Askin's tumor.

In one preferred embodiment, agonistic antibodies of the invention areused to treat Ewing's tumor of bone. In one further referred embodiment,agonistic antibodies of the invention are used to treat peripheralneuroepithelioma.

Antibodies of the present invention may be administered in combinationwith one or more surgical and/or radiological procedures useful in thetreatment of Ewing's sarcoma family tumors.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more surgical and/orradiological procedures useful in the treatment of Ewing's sarcomafamily tumors.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of Ewing'ssarcoma family tumors including, but not limited to, Cyclophosphamide(Cytoxan®, Neosar®, CTX), Doxorubicin (Adriamycin®, Doxil®, Rubex®),Etoposide phosphate (Etopophos®), Etoposide (VP-16, Vepesid®),Filgrastim (Neupogen®, G-CSF), Ifosfamide (IFEX®), Topotecan (Hycamtin®,SK&F-104864, NSC-609699, Evotopin®), and Vincristine (Oncovin®, OncoTCS®, VCR, Leurocristine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofEwing's sarcoma family tumors.

Preferred combinations of therapeutic agents useful in the treatment ofEwing's sarcoma family tumors which may be administered in combinationwith antibodies of the present invention include, but are not limitedto, Cyclophosphamide+Topotecan,Cyclophosphamide+Doxorubicin+Vincristine,Cyclophosphamide+Doxorubicin+Vincristine, alternating withIfosfamide+Etoposide andcyclophosphamide+Doxorubicin+Vincristine,alternating with Filgrastim+Ifosfamide+Etoposide.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of Ewing's sarcoma family tumors.

In further particular embodiments, antibodies of the present inventionare used to treat, ameliorate and/or prevent hematological cancers andpremalignant conditions. Antibodies of the present invention may be usedin combination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or preventhematological cancers and premalignant conditions. Hematological cancerswhich may be treated using antibodies of the present invention include,but are not limited to, non-Hodgkin's lymphoma (e.g., small lymphocyticlymphoma, follicular center cell lymphoma, lymphoplasmacytoid lymphoma,marginal zone lymphoma, mantle cell lymphoma, immunoblastic lymphoma,burkitt's lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma,anaplastic large cell lymphoma and intestinal T-cell lymphoma),leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia andplasma cell neoplasms including multiple myeloma.

In preferred embodiments, agonistic antibodies of the present inventionare used to treat, ameliorate and/or prevent hematological cancers andpremalignant conditions. Agonistic antibodies of the present inventionmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventhematological cancers and premalignant conditions. Hematological cancerswhich may be treated using agonistic antibodies of the present inventioninclude, but are not limited to, non-Hodgkin's lymphoma (e.g., smalllymphocytic lymphoma, follicular center cell lymphoma,lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle celllymphoma, immunoblastic lymphoma, burkitt's lymphoma, lymphoblasticlymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma andintestinal T-cell lymphoma), leukemia, acute lymphocytic leukemia,chronic lymphocytic leukemia and plasma cell neoplasms includingmultiple myeloma.

In one preferred embodiment, agonistic antibodies of the invention areused to treat plasma cell neoplasms. In a specific embodiment, thatplasma cell neoplasm is multiple myeloma.

In another preferred embodiment, agonistic antibodies of the inventionare used to treat non-Hodgkin's lymphoma.

In another preferred embodiment, agonistic antibodies of the inventionare used to treat leukemia. In a specific embodiment, that leukemia isacute lymphocytic leukemia. In another specific embodiment, thatleukemia is chronic lymphocytic leukemia.

Antibodies of the present invention may be administered in combinationwith one or more surgical and/or radiological procedures useful in thetreatment of hematological cancer and premalignant conditions including,but not limited to, bone marrow transplantation, external beam radiationand total body irradiation.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more surgical and/orradiological procedures useful in the treatment of hematological cancerand premalignant conditions including, but not limited to, bone marrowtransplantation, external beam radiation and total body irradiation.

In one preferred embodiment, agonistic antibodies of the presentinvention may be administered in combination with one or more surgicaland/or radiological procedures useful in the treatment of multiplemyeloma including, but not limited to, allogeneic bone marrowtransplantation and peripheral stem cell support.

In another preferred embodiment, agonistic antibodies of the presentinvention may be administered in combination with one or more surgicaland/or radiological procedures useful in the treatment of non-Hodgkin'slymphoma including, but not limited to, allogeneic bone marrowtransplantation and peripheral stem cell support.

In further specific embodiments, agonistic antibodies of the presentinvention may be administered in combination with one or more surgicaland/or radiological procedures useful in the treatment of leukemiaincluding, but not limited to, allogeneic bone marrow transplantationand peripheral stem cell support. In one specific preferred embodiment,agonistic antibodies of the invention are used to treat acutelymphocytic leukemia (ALL). In another specific preferred embodiment,agonistic antibodies of the invention are used to treat chroniclymphocytic leukemia (CLL).

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of multiplemyeloma including, but not limited to, Alkylating agents,Anthracyclines, Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®),Cyclophosphamide (Cytoxan®, Neosar®, CTX), Dexamethasone (Decadron®),Doxorubicin (Adriamycin®, Doxil®, Rubex®), Melphalan (L-PAM, Alkeran®,Phenylalanine mustard), Prednisone, Thalidomide and thalidomide analogs,including but not limited to, lenalidomide (CC-5013, REVLIMID®) andCC-4047 (ACTIMID™), and Vincristine (Oncovorin®, Onco TCS®, VCR,Leurocristine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofmultiple myeloma.

Preferred combinations of therapeutic agents useful in the treatment ofmultiple myeloma which may be administered in combination withantibodies of the present invention include, but are not limited to,Cyclophosphamide+Prednisone, Melphalan+Prednisone (MP),Vincristine+Adriamycin®+Dexamethasone (VAD),Vincristine+Carmustine+Melphalan+Cyclophosphamide+Prednisone (VBMCP; theM2 protocol), and Vincristine+Melphalan+Cyclophosphamide+Prednisonealternating with Vincristine+Carmustine+Doxorubicin+Prednisone(VMCP/VBAP).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of multiple myeloma.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment ofnon-Hodgkin's lymphoma including, but not limited to,2-chlorodeoxyadenosine, Amifostine (Ethyol®, Ethiofos®, WR-272),Bexarotene (Targretin®, Targretin Gel®, Targretin Oral®, LGD1069),Bleomycin (Blenoxane®), Busulfan (Busulfex®, Myleran®), Carboplatin(Paraplatin®, CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®),Chloranbucil (Leukeran®), Cisplatin (Platinol®, CDDP), Cladribine(2-CdA, Leustatin®), Cyclophosphamide (Cytoxan®, Neosar®, CTX),Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®),Dacarbazine (DTIC), Daunorubicin (Daunomycin, DaunoXome®, Daunorubicin®,Cerubidine®), Denileukin diftitox (Ontak®), Dexamethasone (Decadron®),Dolasetron mesylate (Anzemet®), Doxorubicin (Adriamycin®, Doxil®,Rubex®), Erythropoietin (EPO®, Epogen®, Procrit®), Etoposide phosphate(Etopophos®), Etoposide (VP-16, Vepesid®), Fludarabine (Fludara®, FAMP),Granisetron (Kytril®), Hydrocortisone, Idarubicin (Idamycin®, DMDR,IDA), Ifosfamide (IFEX®), Interferon alpha (Alfaferone®, Alpha-IF®),Interferon alpha 2a (Intron A®), Mechlorethamine (Nitrogen Mustard, HN₂,Mustargen®), Melphalan (L-PAM, Alkeran®, Phenylalanine mustard),Methotrexate® (MTX, Mexate®, Folex®), Methylprednisolone (Solumedrol®),Mitoxantrone (Novantrone®, DHAD), Ondansetron (Zofran®), Pentostatin(Nipent®, 2-deoxycoformycin), Perfosfamide(4-hydroperoxycyclophosphamide, 4-HC), Prednisone, Procarbazine(Matulane®), Rituximab® (Rituxan®, anti-CD20 MAb), Thiotepa(triethylenethiophosphaoramide, Thioplex®), Topotecan (Hycamtin®,SK&F-104864, NSC-609699, Evotopin®), Vinblastine (Velban®, VLB),Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®) and Vindesine(Eldisine®, Fildesin®)).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofnon-Hodgkin's lymphoma.

Preferred combinations of therapeutic agents useful in the treatment ofnon-Hodgkin's lymphoma which may be administered in combination withantibodies of the present invention include, but are not limited to,Adriamycin®+Blenoxane+Vinblastine+Dacarbazine (ABVD), Anti-idiotypetherapy (BsAb)+Interferon alpha, Anti-idiotype therapy(BsAb)+Chlorambucil, Anti-idiotype therapy (BsAb)+Interleukin-2, BCNU(Carmustine)+Etoposide+Ara-C (Cytarabine)+Melphalen (BEAM),Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+Vincristine+Procarbazine+Prednisone(BEACOPP), Bryostatin+Vincristine, Cyclophosphamide+BCNU(Carmustine)+VP-16 (Etoposide) (CBV),Cyclophosphamide+Vincristine+Prednisone (CVP),Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine(Oncovorin)+Prednisone (CHOP), Cyclophosphamide+Novantrone®(Mitoxantrone)+Vincristine (Oncovorin)+Prednisone (CNOP),Cyclophosphamide+Doxorubicin+Teniposide+Prednisone,Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine(Oncovorin)+Prednisone+Rituximab (CHOP+Rituximab),Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon alpha,Cytarabine+Bleomycin+Vincristine+Methotrexate (CytaBOM),Dexamethasone+Cytarabine+Cisplatin (DHAP),Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE),Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+Prednisone(Stanford V), Etoposide+Vinblastine+Adriamycin (EVA),Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP),Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC), Fludarabine,Mitoxantrone+Dexamethasone (FMD), Fludarabine, Dexamethasone, Cytarabine(ara-C), +Cisplatin (Platinol®) (FluDAP), Ifosfamide+Cisplatin+Etoposide(ICE), Mechlorethamine+Oncovin® (Vincristine)+Procarbazine+Prednisone(MOPP), Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate withleucovorinrescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone(m-BACOD), Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide(ProMACE), Thiotepa+Busulfan+Cyclophosphamide,Thiotepa+Busulfan+Melphalan, Topotecan+Paclitaxel, and Vincristine(Oncovin®)+Adriamycin®+Dexamethasone (VAD).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of non-Hodgkin's lymphoma.

Further examples of therapeutic agents useful in the treatment ofnon-Hodgkin's lymphoma which may be administered in combination withantibodies of the present invention include, but are not limited to,A007 (4-4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone), AG-2034(AG-2024, AG-2032, GARFT [glycinamide ribonucleoside transformylase]inhibitor), Aldesleukin (IL-2, Proleukin®), Alemtuzumab (Campath®),Alitretinoin (Panretin®, LGN-1057), Altretamine (Hexylen®,hexamethylmelamine, Hexastat®), Aminocamptothecin (9-AC,9-Aminocamptothecin, NSC 603071), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv,anti-NHL MAb), Anti-idiotype therapy (BsAb), Arabinosylguanine (Ara-G,GW506U78), Arsenic trioxide (Trisenox®, ATO), B43-Genistein (anti-CD19Ab/genistein conjugate), B7 antibody conjugates, Betathine (Beta-LT),BLyS antagonists, Bryostatin-1 (Bryostatin®, BMY-45618, NSC-339555),CHML (Cytotropic Heterogeneous Molecular Lipids), Clofarabine(chloro-fluoro-araA), Daclizumab (Zenapax®), Depsipeptide (FR901228,FK228), Dolastatin-10 (DOLA-10, NSC-376128), Epirubicin (Ellence®, EPI,4′ epi-doxorubicin), Epratuzumab (Lymphocide®, humanized anti-CD22,HAT), Fly3/flk2 ligand (Mobista®), G3139 (Genasense®, GentaAnticode®,Bcl-2 antisense), HuID10 (anti-HLA-DR MAb, SMART ID10), HumaLYM(anti-CD20 MAb), Ibritumomab tiuxetan (Zevalin®), Interferon gamma(Gamma-interferon, Gamma 1000, Gamma-IF), Irinotecan (Camptosar®,CPT-11, Topotecin®, CaptoCPT-1), ISIS-2053, ISIS-3521 (PKC-alphaantisense), Lmb-2 immunotoxin (anti-CD25 recombinant immuno toxin,anti-Tac(Fv)-PE38), Leuvectin® (cytofectin+IL-2 gene, IL-2 genetherapy), Lym-1 (131-I LYM-I), Lymphoma vaccine (Genitope), Nelarabine(Compound 506, U78), Neugene compounds (Oncomyc-NG®, Resten-NG®, mycantisense), NovoMAb-G2 scFv (NovoMAb-G2 IgM), 06-benzylguanine (BG,Procept®), Oxaliplatin (Eloxatine®, Eloxatin®), Paclitaxel (Paxene®,Taxol®), Paclitaxel-DHA (Taxoprexin®), Peldesine (BCX-34, PNPinhibitor), Rebeccamycin and Rebeccamycin analogues, SCH-66336,Sobuzoxane (MST-16, Perazolin®), SU5416 (Semaxanib®, VEGF inhibitor),TER-286, Thalidomide and thalidomide analogs, including but not limitedto, lenalidomide (CC-5013, REVLIMID®) and CC-4047 (ACTIMID™), TNP-470(AGM-1470), Tositumomab (Bexxar®), Valspodar (PSC 833), Vaxid (B-celllymphoma DNA vaccine), Vinorelbine (Navelbine®), WF10 (macrophageregulator) and XR-9576 (XR-9351, P-glycoprotein/MDR inhibitor).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofnon-Hodgkin's lymphoma.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of acutelymphocytic leukemia including, but not limited to, Amsacrine,Carboplatin (Paraplatin®, CBDCA), Carmustine (DTI-015, BCNU, BiCNU,Gliadel Wafer®), Cholecaliferol, Cyclophosphamide (Cytoxan®, Neosar®,CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®),Daunorubicin (Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®),Dexamethasone (Decadron®), Doxorubicin (Adriamycin®, Doxil®, Rubex®),Etoposide (VP-16, Vepesid®), Filgrastam® (Neupogen®, G-CSF, Leukine®),Fludarabine (Fludara®, FAMP), Idarubicin (Idamycin®, DMDR, IDA),Ifosfamide (IFEX®), Imatinib mesylate (STI-571, Imatinib®, Glivec®,Gleevec®, Ab1 tyrosine kinase inhibitor), Interferon gamma(Gamma-interferon, Gamma 1000, Gamma-IF), L-asparaginase (Elspar®,Crastinin®, Asparaginase Medac®, Kidrolase®), Mercaptopurine(6-mercaptopurine, 6-MP), Methotrexate® (MTX, Mexate®, Folex®),Mitoxantrone (Novantrone®, DHAD), Pegaspargase® (Oncospar®), Prednisone,Retinoic acid, Teniposide (VM-26, Vumon®), Thioguanine (6-thioguanine,6-TG), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®),Tretinoin (Retin-A®, Atragen®, ATRA, Vesanoid®) and Vincristine(Oncovorin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofacute lymphocytic leukemia.

Further examples of therapeutic agents useful in the treatment of acutelymphocytic leukemia which may be administered in combination withantibodies of the present invention include, but are not limited to,Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminopterin,Annamycin (AR-522, annamycin LF, Aronex®), Arabinosylguanine (Ara-G,GW506U78, Nelzarabine®), Arsenic trioxide (Trisenox®, ATO, Atrivex®V),B43-Genistein (anti-CD19 Ab/genistein conjugate), B43-PAP (anti-CD19Ab/pokeweed antiviral protein conjugate), Cordycepin, CS-682, Decitabine(5-aza-2′-deoxyytidine), Dolastatin-10 (DOLA-10, NSC-376128), G3139(Genasense®, GentaAnticode®, Bcl-2 antisense), Irofulven (MGI-114,Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Quinine,TNP-470 (AGM-1470, Fumagillin), Trimetrexate (Neutrexin®), Troxacitabine(BCH-204, BCH-4556, Troxatyl®), UCN-01 (7-hydroxystaurosporine),WHI-P131 and WT1 Vaccine.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofacute lymphocytic leukemia.

Preferred combinations of therapeutic agents useful in the treatment ofacute lymphocytic leukemia which may be administered in combination withantibodies of the present invention include, but are not limited to,Carboplatin+Mitoxantrone, Carmustine+Cyclophosphamide+Etoposide,Cytarabine+Daunorubicin, Cytarabine+Doxorubicin, Cytarabine+Idarubicin,Cytarabine+Interferon gamma, Cytarabine+L-asparaginase,Cytarabine+Mitoxantrone, Cytarabine+Fludarabine and Mitoxantrone,Etoposide+Cytarabine, Etoposide+Ifosfamide, Etoposide+Mitoxantrone,Ifosfamide+Etoposide+Mitoxantrone, Ifosfamide+Teniposide,Methotrexate+Mercaptopurine,Methotrexate+Mercaptopurine+Vincristine+Prednisone,Phenylbutyrate+Cytarabine, Phenylbutyrate+Etoposide,Phenylbutyrate+Topotecan, Phenylbutyrate+Tretinoin, Quinine+Doxorubicin,Quinine+Mitoxantrone+Cytarabine, Thioguanine+Cytarabine+Amsacrine,Thioguanine+Etoposide+Idarubicin, Thioguanine+Retinoicacid+Cholecaliferol, Vincristine+Prednisone, Vincristine+Prednisone andL-asparaginase,Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin,Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Filgrastim,Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate,andVincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate+Filgrastim.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of acute lymphocytic leukemia.

Antibodies of the present invention may be administered in combinationwith one or more therapeutic agents useful in the treatment of chroniclymphocytic leukemia including, but not limited to, Chlorambucil(Leukeran®), Cladribine (2-CdA, Leustatin®), Cyclophosphamide (Cytoxan®,Neosar®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside,DepoCyt®, cytarabine ocfosfate, ara-CMP), Doxorubicin (Adriamycin®,Doxil®, Rubex®), Fludarabine (Fludara®, FAMP), Pentostatin (Nipent®,2-deoxycoformycin), Prednisone and Vincristine (Oncovorin®, Onco TCS®,VCR, Leurocristine®).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofchronic lymphocytic leukemia.

Further examples of therapeutic agents useful in the treatment ofchronic lymphocytic leukemia which may be administered in combinationwith antibodies of the present invention include, but are not limitedto, Alemtuzumab (Campath®), Aminocamptothecin (9-AC,9-Aminocamptothecin, NSC 603071), Aminopterin, Annamycin (AR-522,annamycin LF, Aronex®), Arabinosylguanine (Ara-G, GW506U78,Nelzarabine®, Compound 506U78), Arsenic trioxide (Trisenox®, ATO,Atrivex®), Bryostatin-1 (Bryostatin®, BMY-45618, NSC-339555), CS-682,Dolastatin-10 (DOLA-10, NSC-376128), Filgrastim (Neupogen®, G-CSF,Leukine), Flavopiridol (NSC-649890, HMR-1275), G3139 (Genasense®,GentaAnticode®, Bcl-2 antisense), Irofulven (MGI-114, Ivofulvan,Acylfulvene analogue), MS-209, Phenylbutyrate, Rituximab® (Rituxan®,anti-CD20 MAb), Thalidomide and thalidomide analogs, including but notlimited to, lenalidomide (CC-5013, REVLIMID®) and CC-4047 (ACTIMID™),Theophylline, TNP-470 (AGM-1470, Fumagillin), UCN-01(7-hydroxystaurosporine) and WHI-P131.

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofchronic lymphocytic leukemia.

Preferred combinations of therapeutic agents useful in the treatment ofchronic lymphocytic leukemia which may be administered in combinationwith antibodies of the present invention include, but are not limitedto, Fludarabine+Prednisone, andCyclophosphamide+Doxorubicin+Vincristine+Prednisone (CHOP).

In preferred embodiments, agonistic antibodies of the invention areadministered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of chronic lymphocytic leukemia.

Anti-DR4 antibodies may be utilized in combination with other monoclonalor chimeric antibodies, or with lymphokines, tumor necrosis factors orTNF-related molecules (e.g., TNF-α, TNF-β, TNF-γ, TNF-γ-β, TNF-γ-β, andTRAIL), or hematopoietic growth factors (e.g., IL-2, IL-3 and IL-7). Forexample, agonistic anti-DR4 antibodies may be administered inconjunction with TRAIL when one seeks to induce DR4 mediated cell deathin cells which express DR4 receptors of the invention. Combinationtherapies of this nature, as well as other combination therapies, arediscussed below in more detail.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M,10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹¹M, 5×10⁻¹¹M,10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M,and 10⁻¹⁵M.

Polypeptide Assays

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of DR4 protein,or the soluble form thereof, in cells and tissues, includingdetermination of normal and abnormal levels. Thus, for instance, adiagnostic assay in accordance with the invention for detectingover-expression of DR4, or soluble form thereof, compared to normalcontrol tissue samples may be used to detect the presence of tumors, forexample. Assay techniques that can be used to determine levels of aprotein, such as a DR4 protein of the present invention, or a solubleform thereof, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Assaying DR4 protein levels in a biological sample can occur using anyart-known method. By “biological sample” is intended any biologicalsample obtained from an individual, cell line, tissue culture, or othersource containing DR4 receptor protein or mRNA. Preferred for assayingDR4 protein levels in a biological sample are antibody-based techniques.For example, DR4 protein expression in tissues can be studied withclassical immunohistological methods. (Jalkanen, M., et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingDR4 protein gene expression include immunoassays, such as the enzymelinked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels, such asglucose oxidase, radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon(¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(^(99m)Tc), and fluorescent labels, such as fluorescein and rhodamine,and biotin.

Transgenics and “Knock-Outs”

The proteins of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., nucleic acids of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pluripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety. Further, the contents of each of the documentsrecited in this paragraph is herein incorporated by reference in itsentirety. See also, U.S. Pat. No. 5,464,764 (Capecchi et al.,Positive-Negative Selection Methods and Vectors); U.S. Pat. No.5,631,153 (Capecchi et al., Cells and Non-Human Organisms ContainingPredetermined Genomic Modifications and Positive-Negative SelectionMethods and Vectors for Making Same); U.S. Pat. No. 4,736,866 (Leder etal., Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagneret al., Genetic Transformation of Zygotes); each of which is herebyincorporated by reference in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campbell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Science 265:103-106 (1994)). The regulatorysequences required for such a cell-type specific inactivation willdepend upon the particular cell type of interest, and will be apparentto those of skill in the art. The contents of each of the documentsrecited in this paragraph is herein incorporated by reference in itsentirety.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of DR4 polypeptides, studyingconditions and/or disorders associated with aberrant DR4 expression, andin screening for compounds effective in ameliorating such conditionsand/or disorders.

In further embodiments of the invention, cells that are geneticallyengineered to express the proteins of the invention, or alternatively,that are genetically engineered not to express the proteins of theinvention (e.g., knockouts) are administered to a patient in vivo. Suchcells may be obtained from the patient (i.e., animal, including human)or an MHC compatible donor and can include, but are not limited tofibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),adipocytes, muscle cells, endothelial cells, etc. The cells aregenetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally. Alternatively, the cells can be incorporated into amatrix and implanted in the body, e.g., genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a lymphatic orvascular graft. (See, for example, Anderson et al. U.S. Pat. No.5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of whichis incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well-known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form that, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Therapeutics

The Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes(Goeddel, D. V. et al., “Tumor Necrosis Factors: Gene Structure andBiological Activities,” Symp. Quant. Biol. 51:597-609 (1986), ColdSpring Harbor; Beutler, B., and Cerami, A., Annu. Rev. Biochem.57:505-518 (1988); Old, L. J., Sci. Am. 258:59-75 (1988); Fiers, W.,FEBS Lett. 285:199-224 (1991)). The TNF-family ligands induce suchvarious cellular responses by binding to TNF-family receptors, includingthe DR4 receptors of the present invention.

DR4 polynucleotides and polypeptides of the invention may be used indeveloping treatments and/or prevention methods for any disordermediated (directly or indirectly) by defective, or insufficient amountsof DR4. DR4 polypeptides may be administered to a patient (e.g., mammal,preferably human) afflicted with such a disorder. Alternatively, a genetherapy approach may be applied to treat and/or prevent such disorders.Disclosure herein of DR4 nucleotide sequences permits the detection ofdefective DR4 genes, and the replacement thereof with normalDR4-encoding genes. Defective genes may be detected in in vitrodiagnostic assays, and by comparison of the DR4 nucleotide sequencedisclosed herein with that of a DR4 gene derived from a patientsuspected of harboring a defect in this gene. Defective genes may bereplaced with normal DR4-encoding genes using techniques known to oneskilled in the art.

In another embodiment, the polypeptides of the present invention areused as a research tool for studying the biological effects that resultfrom inhibiting TRAIL/DR4 interactions on different cell types. DR4polypeptides also may be employed in in vitro assays for detecting TRAILor DR4 or the interactions thereof.

In another embodiment, a purified DR4 polypeptide or DR4 antagonist ofthe invention is used to inhibit binding of TRAIL to endogenous cellsurface TRAIL receptors. Certain ligands of the TNF family (of whichTRAIL is a member) have been reported to bind to more than one distinctcell surface receptor protein. TRAIL likewise is believed to bindmultiple cell surface proteins. By binding TRAIL, soluble DR4polypeptides and/or DR4 antagonists of the present invention may beemployed to inhibit the binding of TRAIL not only to cell surface DR4,but also to TRAIL receptor proteins that are distinct from DR4. Thus, inanother embodiment, a DR4 polypeptide and/or antagonist is used toinhibit a biological activity of TRAIL, in in vitro or in vivoprocedures. By inhibiting binding of TRAIL to cell surface receptors, aDR4 polypeptide and antagonist also inhibits biological effects thatresult from the binding of TRAIL to endogenous receptors. Various formsof DR4 polypeptides may be employed, including, for example, theabove-described DR4 fragments, derivatives, and variants that arecapable of binding TRAIL. In a preferred embodiment, a soluble DR4 isemployed to inhibit a biological activity of TRAIL, e.g., to inhibitTRAIL-mediated apoptosis of cells susceptible to such apoptosis.

In a further embodiment, DR4 compositions (e.g., DR4 polynucleotides,polypeptides, agonists and/or antagonists) of the invention areadministered to a mammal (e.g., a human) to treat and/or prevent aTRAIL-mediated disorder. Such TRAIL-mediated disorders includeconditions caused (directly or indirectly) or exacerbated by TRAIL.

Cells which express the DR4 polypeptide and are believed to have apotent cellular response to DR4 ligands include amniotic cells, heart,liver cancer, kidney, peripheral blood leukocytes, activated T-cells,tissue corresponding to Th2 cells, human tonsils, and CD34 depletedbuffy coat (cord blood). By “a cellular response to a TNF-family ligand”is intended any genotypic, phenotypic, and/or morphologic change to acell, cell line, tissue, tissue culture or patient that is induced by aTNF-family ligand. As indicated, such cellular responses include notonly normal physiological responses to TNF-family ligands, but alsodiseases associated with increased apoptosis or the inhibition ofapoptosis. Apoptosis-programmed cell death-is a physiological mechanisminvolved in the deletion of peripheral T lymphocytes of the immunesystem, and its dysregulation can lead to a number of differentpathogenic processes (Ameisen, J. C., AIDS 8:1197-1213 (1994); Krammer,P. H. et al., Curr. Opin. Immunol. 6:279-289 (1994)).

Diseases associated with increased cell survival, or the inhibition ofapoptosis, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostrate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), information graft v. host disease, acute graft rejection,and chronic graft rejection. In preferred embodiments, DR4polynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metastasis of cancers, inparticular those listed above or in the paragraph that follows.

Additional diseases or conditions associated with increased cellsurvival include, but are not limited to, progression, and/or metastasesof malignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Additional diseases or conditions associated with increased cellsurvival include, but are not limited to, premalignant conditions. Suchconditions are known or suspected of preceding progression to neoplasiaor cancer, in particular, where non-neoplastic cell growth consisting ofhyperplasia, metaplasia, or most particularly, dysplasia has occurred(for review of such abnormal growth conditions, see Robbins and Angell,1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp.68-79.)

Hyperplasia is a form of controlled cell proliferation, involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. Hyperplastic disorders which can bediagnosed, prognosed, prevented, and/or treated with compositions of theinvention (including polynucleotides, polypeptides, agonists orantagonists) include, but are not limited to, angiofollicularmediastinal lymph node hyperplasia, angiolymphoid hyperplasia witheosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia,benign giant lymph node hyperplasia, cementum hyperplasia, congenitaladrenal hyperplasia, congenital sebaceous hyperplasia, cystichyperplasia, cystic hyperplasia of the breast, denture hyperplasia,ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia,focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibroushyperplasia, inflammatory papillary hyperplasia, intravascular papillaryendothelial hyperplasia, nodular hyperplasia of prostate, nodularregenerative hyperplasia, pseudoepitheliomatous hyperplasia, senilesebaceous hyperplasia, and verrucous hyperplasia.

Metaplasia is a form of controlled cell growth in which one type ofadult or fully differentiated cell substitutes for another type of adultcell. Metaplastic disorders which can be diagnosed, prognosed,prevented, and/or treated with compositions of the invention (includingpolynucleotides, polypeptides, agonists or antagonists) include, but arenot limited to, agnogenic myeloid metaplasia, apocrine metaplasia,atypical metaplasia, autoparenchymatous metaplasia, connective tissuemetaplasia, epithelial metaplasia, intestinal metaplasia, metaplasticanemia, metaplastic ossification, metaplastic polyps, myeloidmetaplasia, primary myeloid metaplasia, secondary myeloid metaplasia,squamous metaplasia, squamous metaplasia of amnion, and symptomaticmyeloid metaplasia.

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation. Dysplastic disorders which can be diagnosed,prognosed, prevented, and/or treated with compositions of the invention(including polynucleotides, polypeptides, agonists or antagonists)include, but are not limited to, anhidrotic ectodermal dysplasia,anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigitaldysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervicaldysplasia, chondroectodermal dysplasia, cleidocranial dysplasia,congenital ectodermal dysplasia, craniodiaphysial dysplasia,craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentindysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia,encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,epithelial dysplasia, faciodigitogenital dysplasia, familial fibrousdysplasia of jaws, familial white folded dysplasia, fibromusculardysplasia, fibrous dysplasia of bone, florid osseous dysplasia,hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammarydysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondinidysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia,multiple epiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders include, but are not limited to,benign dysproliferative disorders (e.g., benign tumors, fibrocysticconditions, tissue hypertrophy, intestinal polyps, colon polyps, andesophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer'sSkin, solar cheilitis, and solar keratosis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents and/or procedures in the treatment,prevention, amelioration and/or cure of cancers and premalignantconditions.

In preferred embodiments, agonists and/or antagonists of the inventionmay be administered in combination with one or more therapeutic agentsand/or procedures in the treatment, prevention, amelioration and/or cureof cancers and premalignant conditions.

Therapeutic agents, useful in the treatment, prevention, ameliorationand/or cure of cancers and premalignant conditions, with whichpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered, include, for example,biological agents (e.g., inhibitors of signaling pathways, inhibitors ofgene transcription, inhibitors of multi-drug resistance (MDR)mechanisms, inhibitors of angiogenesis, inhibitors of matrixmetalloproteinases, hormones and hormone antagonists, and compounds ofunknown mechanism), chemotherapeutic agents (e.g., alkylating agents,antimetabolites, farnesyl transferase inhibitors, mitotic spindleinhibitors (plant-derived alkaloids), nucleotide analogs, platinumanalogs, and topoisomerase inhibitors), corticosteroids, gene therapies,immunotherapeutic agents (e.g., monoclonal antibodies, cytokines andvaccines), phototherapy, radiosensitizing agents, treatment supportagents (e.g., anti-emetic agents, analgesic agents and hematopoieticagents), and other miscellaneous drug types. Therapeutic procedures,useful in the treatment, prevention, amelioration and/or cure of cancersand premalignant conditions, with which polynucleotides and/orpolypeptides of the invention and/or agonists and/or antagonists thereofmay be administered, include, for example, but are not limited to,surgical procedures and radiation therapies.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents and/or therapeutic procedures in the treatment,prevention, amelioration and/or cure of cancers and premalignantconditions.

In specific embodiments, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof may be administeredin combination with one or more therapeutic agents useful in thetreatment, prevention, amelioration and/or cure of cancers andpremalignant conditions, including, but not limited to, 81C6(Anti-tenascin monoclonal antibody), 2-chlorodeoxyadenosine, A007(4-4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone), Abarelix((Abarelix-Depot-Me, PPI-149, R-3827); Abiraterone Acetate® (CB-7598,CB-7630), ABT-627 (ET-1 inhibitor), ABX-EGF (anti-EGFr MAb),Acetyldinaline (CI-994, GOE-5549, GOR-5549, PD-130636), AG-2034(AG-2024, AG-2032, GARFT [glycinamide ribonucleoside transformylase]inhibitor), Alanosine, Aldesleukin (IL-2, Proleukin®), Alemtuzumab®(Campath®), Alitretinoin (Panretin®, LGN-1057), Allopurinol (Aloprim®,Zyloprim®), Altretamine (Hexylen®, hexamethylmelamine, Hexastat®),Amifostine (Ethyol®), Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC603071), Aminoglutethimide (Cytadren®), Aminolevulinic acid (Levulan®,Kerastick®), Aminopterin, Amsacrine, Anastrozole (Arimidex®),Angiostatin, Annamycin (AR-522, annamycin LF, Aronex®), Anti-idiotypetherapy (BsAb), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv, anti-NHL MAb),APC-8015 (Provenge®, Dendritic cell therapy), Aplidine (Aplidin®,Aplidina®), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®, Compound506U78), Arsenic trioxide (Trisenox®, ATO, Atrivex®), Avorelin®(Meterelin®, MF-6001, EP-23904), B43-Genistein (anti-CD19 Ab/genisteinconjugate), B43-PAP (anti-CD19 Ab/pokeweed antiviral protein conjugate),B7 antibody conjugates, BAY 43-9006 (Raf kinase inhibitor), BBR 3464,Betathine (Beta-LT), Bevacizumab® (Anti-VEGF monoclonal antibody,rhuMAb-VEGF), Bexarotene (Targretin®, LGD1069), BIBH-1 (Anti-FAP MAb),BIBX-1382, Biclutamide (Casodex®), Biricodar dicitrate (Incel®, IncelMDR Inhibitor), Bleomycin (Blenoxane®), BLP-25 (MUC-1 peptide), BLySantagonists, BMS-214662 (BMS-192331, BMS-193269, BMS-206635), BNP-1350(BNPI-1100, Karenitecins), Boronated Protoporphyrin Compound (PDIT,Photodynamic Immunotherapy), Bryostatin-1 (Bryostatin®, BMY-45618,NSC-339555), Budesonide (Rhinocort®), Busulfan (Busulfex®, Myleran®),C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb, Cetuximab®), C242-DM1(buC242-DM1), Cabergoline (Dostinex®), Capecitabine (Xeloda®,Doxifluridine®, oral 5-FU), Carbendazin® (FB-642), Carboplatin(Paraplatin®), CBDCA), Carboxyamidotriazole (NSC 609974, CAI, L-651582),Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), CC49-zeta genetherapy, CEA-cide® (Labetuzumab®, Anti-CEA monoclonal antibody, hMN-14),CeaVac® (MAb 3H1), Celecoxib (Celebrex®), CEP-701 (KT-5555), Cereport®(Lobradimil®, RMP-7), Chlorambucil (Leukeran®), CHML (CytotropicHeterogeneous Molecular Lipids), Cholecaliferol, CI-1033 (Pan-erbB RTKinhibitor), Cilengitide (EMD-121974, integrin alphavbeta3 antagonist),Cisplatin (Platinol®, CDDP), Cisplatin-epinephrine gel (IntraDose®,FocaCist®), Cisplatin-liposomal (SPI-077), 9-cis retinoic acid (9-cRA),Cladribine (2-CdA, Leustatin®), Clofarabine (chloro-fluoro-araA),Clonadine hydrochloride (Duraclon®), CMB-401 (Anti-PEMMAb/calicheamycin), CMT-3 (COL-3, Metastat®), Cordycepin, Cotara®(chTNT-1/B, [¹³¹I]-chTNT-1/B), CN-706, CP-358774 (Tarceva®, OSI-774,EGFR inhibitor), CP-609754, CP IL-4-toxin (IL-4 fusion toxin), CS-682,CT-2584 (Apra®, CT-2583, CT-2586, CT-3536), CTP-37 (Avicine®, hCGblocking vaccine), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine(Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®), D-limonene,DAB389-EGF (EGF fusion toxin), Dacarbazine (DTIC), Daclizumab®(Zenapax®), Dactinomycin (Cosmegen®), Daunomycin (Daunorubicin®,Cerubidine®), Daunorubicin (DaunoXome®, Daunorubicin®, Cerubidine®V),DeaVac® (CEA anti-idiotype vaccine), Decitabine (5-aza-2′-deoxyytidine),Declopramide (Oxi-104), Denileukin diftitox (Ontak®), Depsipeptide(FR901228, FK228), Dexamethasone (Decadron®), Dexrazoxane (Zinecard®),Diethylnorspermine (DENSPM), Diethylstilbestrol (DES),Dihydro-5-azacytidine, Docetaxel (Taxotere®, Taxane®), Dolasetronmesylate (Anzemet®), Dolastatin-10 (DOLA-10, NSC-376128), Doxorubicin(Adriamycin®, Doxil®, Rubex®), DPPE, DX-8951f (DX-8951), Edatrexate,EGF-P64k Vaccine, Elliott's B Solution®, EMD-121974, Endostatin,Eniluracil (776c85), EO9 (EO1, EO4, EO68, EO70, EO72), Epirubicin(Ellence®, EPI, 4′ epi-doxorubicin), Epratuzumab® (Lymphocide®,humanized anti-CD22, HAT), Erythropoietin (EPO®, Epogen®, Procrit®),Estramustine (Emcyt®), Etanidazole (Radinyl®), Etoposide phosphate(Etopophos®), Etoposide (VP-16, Vepesid®), Exemestane (Aromasin®,Nikidess®), Exetecan mesylate (DX-8951, DX-8951f, Exisulind (SAAND,Aptosyn®, cGMP-PDE2 and 5 inhibitor), F19 (Anti-FAP monoclonal antibody,iodinated anti-FAP MAb), Fadrozole (Afema®, Fadrozole hydrochloride,Arensin®), Fenretinide® (4HPR), Fentanyl citrate (Actiq®), Filgrastim(Neupogen®, G-CSF), FK-317 (FR-157471, FR-70496), Flavopiridol(HMR-1275), Fly3/flk2 ligand (Mobista®), Fluasterone, Fludarabine(Fludara®, FAMP), Fludeoxyglucose (F-18®), Fluorouracil (5-FU, Adrucil®,Fluoroplex®, Efudex®), Flutamide (Eulexin®), FMdC (KW-2331, MDL-101731),Formestane (Lentaron®), Fotemustine (Muphoran®, Mustophoran®), FUDR(Floxuridine®), Fulvestrant (Faslodex®), G3139 (Genasense®,GentaAnticode®, Bcl-2 antisense), Gadolinium texaphyrin (Motexafingadolinium, Gd-Tex®, Xcytrin®), Galarubicin hydrochloride (DA-125),GBC-590, Gastrimmune® (Anti-gastrin-17 immunogen, anti-g17), Gemcitabine(Gemto®, Gemzar®), Gentuzumab-ozogamicin (Mylotarg®), GL331, Globo Hhexasaccharide (Globo H-KLH®), Glufosfamide® (β-D-glucosyl-isofosfamidemustard, D19575, INN), Goserelin acetate (Zoladex®), Granisetron(Kytril®), GVAX (GM-CSF gene therapy), Her-2/Neu vaccine, Herceptin®(Trastuzumab®, Anti-HER-2 monoclonal antibody, Anti-EGFR-2 MAb),HSPPC-96 (HSP cancer vaccine, gp96 heat shock protein-peptide complex),Hu1D10 (anti-HLA-DR MAb, SMART 1D10), HumaLYM (anti-CD20 MAb),Hydrocortisone, Hydroxyurea (Hydrea®), Hypericin® (VIMRxyn®), I-131Lipidiol®, Ibritumomab® tiuxetan (Zevalin®), Idarubicin (Idamycin®,DMDR, IDA), Ifosfamide (IFEX®), Imatinib mesylate (STI-571, Imatinib®,Glivec®, Gleevec®, Ab1 tyrosine kinase inhibitor), INGN-101 (p53 genetherapy/retrovirus), INGN-201 (p53 gene therapy/adenovirus), Interferonalpha (Alfaferone®, Alpha-IF®), Interferon alpha 2a (Intron A®),Interferon gamma (Gamma-interferon, Gamma 100®, Gamma-IF), Interleukin-2(ProleiukinR®), Intoplicine (RP 60475), Irinotecan (Camptosar®, CPT-11,Topotecin®V, CaptoCPT-1), Irofulven (MGI-114, Ivofulvan, Acylfulveneanalogue), ISIS-2053 (PKC-alpha antisense), ISIS-2503 (Ras antisense),ISIS-3521 (PKC-alpha antisense), ISIS-5132 (K-ras/raf antisense),Isotretinoin (13-CRA, 13-cis retinoic acid, Accutane®), Ketoconazole(Nizoral®), KRN-8602 (MX, MY-5, NSC-619003, MX-2), L-778123 (Rasinhibitors), L-asparaginase (Elspar®, Crastinin®, Asparaginase Medac®,Kidrolase®), Leflunomide (SU-101, SU-0200), Letrozole (Femara®),Leucovorin (Leucovorin®, Welicovorin®), Leuprolide acetate (Viadur®,Lupron®, Leuprogel®, Eligard®), Leuvectin® (cytofectin+IL-2 gene, IL-2gene therapy), Levamisole (Ergamisol®), Liarozole (Liazal, Liazol,R-75251, R-85246, Ro-85264), Lmb-2 immunotoxin (anti-CD25 recombinantimmuno toxin, anti-Tac(Fv)-PE38), Lometrexol (T-64, T-904064), Lomustine(CCNU®, CeeNU®), LY-335979, Lym-1 (131-I LYM-1), Lymphoma vaccine(Genitope), Mannan-MUC1 vaccine, Marimastat® (BB-2516, TA-2516, MMPinhibitor), MDX-447 (MDX-220, BAB-447, EMD-82633, H-447,anti-EGFr/FcGammaR1r), Mechlorethamine (Nitrogen Mustard, HN₂,Mustargen®), Megestrol acetate (Megace®, Pallace®), Melphalan (L-PAM,Alkeran®, Phenylalanine mustard), Mercaptopurine (6-mercaptopurine,6-MP), Mesna (Mesnex®), Methotrexate® (MTX, Mexate®, Folex®),Methoxsalen (Uvadex®), 2-Methoxyestradiol (2-ME, 2-ME2),Methylprednisolone (Solumedrol®), Methyltestosterone (Android-10®,Testred®, Virilon®), MGV, Mitomycin C (Mitomycin®, Mutamycin®, MitoExtra®), Mitoxantrone (Novantrone®, DHAD), Mitumomab® (BEC-2,EMD-60205), Mivobulin isethionate (CI-980), MN-14 (Anti-CEAimmunoradiotherapy, ¹³¹I-MN-14, ¹⁸⁸Re-MN-14), Motexafin Lutetium(Lutrin®, Optrin®, Lu-Tex®, lutetium texaphyrin, Lucyn®, Antrin®),MPV-2213ad (Finrozole®), MS-209, Muc-1 vaccine, NaPro Paclitaxel,Nelarabine (Compound 506, U78), Neovastat® (AE-941, MMP inhibitor),Neugene compounds (Oncomyc-NG, Resten-NG, myc antisense), Nilutamide(Nilandron®), NovoMAb-G2 scFv (NovoMAb-G2 IgM), NPI-0052 (proteasomeinhibitor), 06-benzylguanine (BG, Procept®), Octreotide acetate(Sandostatin LAR® Depot), Odansetron (Zofran®R), Onconase (Ranpimase®),OncoVAX-CL, OncoVAX-CL Jenner (GA-733-2 vaccine), OncoVAX-P(OncoVAX-PrPSA), Onyx-015 (p53 gene therapy), Oprelvekin (Neumage®),Orzel (Tegafur+Uracil+Leucovorin), Oxaliplatin (Eloxatine®, Eloxatin®),Pacis® (BCG, live), Paclitaxel (Paxene®, Taxol®), Paclitaxel-DHA(Taxoprexin®), Pamidronate (Aredia®), PC SPES, Pegademase (Adagen®,Pegademase bovine), Pegaspargase® (Oncospar®), Peldesine (BCX-34, PNPinhibitor), Pemetrexed disodium (Alimta®, MTA, multitargeted antifolate,LY 231514), Pentostatin (Nipent®, 2-deoxycoformycin), Perfosfamide(4-hydroperoxycyclophosphamide, 4-HC), Perillyl alcohol (perillaalcohol, perillic alcohol, perillol, NSC-641066), Phenylbutyrate,Pirarubicin (THP), Pivaloyloxymethyl butyrate (AN-9, Pivanex®), Porfimersodium (Photofrin®), Prednisone, Prinomastat® (AG-3340, MMP inhibitor),Procarbazine (Matulane®), PROSTVAC, Providence Portland Medical CenterBreast Cancer Vaccine, PS-341 (LDP-341, 26S proteasome inhibitor), PSMAMAb (Prostate Specific Membrane Antigen monoclonal antibody),Pyrazoloacridine (NSC-366140, PD-115934), Quinine, R115777 (Zarnestra®),Raloxifene hydrochloride (Evista®, Keoxifene hydrochloride), Raltitrexed(Tomudex®, ZD-1694), Rebeccamycin, Retinoic acid, R-flurbiprofen(Flurizan®, E-7869, MPC-7869), RFS-2000 (9-nitrocamptothecan, 9-NC,Rubitecan®), Rituximab® (Rituxan®, anti-CD20 MAb), RSR-13 (GSJ-61),Satraplatin (BMS-182751, JM-216), SCH 6636, SCH-66336, Sizofilan® (SPG,Sizofuran®, Schizophyllan®, Sonifilan®), SKI-2053R(NSC-D644591),Sobuzoxane (MST-16, Perazolin®), Squalamine (MSI-1256F), SR-49059(vasopressin receptor inhibitor, Vla), Streptozocin (Zanosar®), SU5416(Semaxanib®, VEGF inhibitor), SU6668 (PDGF-TK inhibitor), T-67(T-138067, T-607), Talc (Sclerosol®), Tamoxifen (Nolvadex®), Taurolidine(Taurolin®), Temozolamide (Temodar®, NSC 362856), Teniposide (VM-26,Vumon®), TER-286, Testosterone (Andro®, Androderm®, Testoderm TTS®,Testoderm®, Depo-Testosterone®, Androgel®, depoAndro®), Tf-CRM107(Transferrin-CRM-107), Thalidomide and thalidomide analogs, includingbut not limited to, lenalidomide (CC-5013, REVLIMID®) and CC-4047(ACTIMID™), Theratope, Thioguanine (6-thioguanine, 6-TG), Thiotepa(triethylenethiophosphaoramide, Thioplex®), Thymosin alpha I (Zadaxin®,Thymalfasin®), Tiazofurin (Thiazole®), Tirapazamine (SR-259075, SR-4233,Tirazone®, Win-59075), TNP-470 (AGM-1470, Fumagillin), Tocladesine(8-Cl-cAMP), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®),Toremifene (Estrimex®, Fareston®), Tositumomab® (Bexxar®), Tretinoin(Retin-A®, Atragen®, ATRA, Vesanoid®), TriAb® (anti-idiotype antibodyimmune stimulator), Trilostane (Modrefen®), Triptorelin pamoate(Trelstar Depots, Decapeptyl®), Trimetrexate (Neutrexin®), Troxacitabine(BCH-204, BCH-4556, Troxatyl®), TS-1, UCN-01 (7-hydroxystaurosporine),Valrubicin (Valstar®), Valspodar (PSC 833), Vapreotide® (BMY-41606),Vaxid (B-cell lymphoma DNA vaccine), Vinblastine (Velban®, VLB),Vincristine (Oncovin®), Onco TCS®, VCR, Leurocristine®), Vindesine(Eldisine®, Fildesin®V), Vinorelbine (Navelbine®), Vitaxin® (LM-609,integrin alphavbeta3 antagonistic MAb), WF10 (macrophage regulator),WHI-P131, WT1 Vaccine, XR-5000 (DACA), XR-9576 (XR-9351,P-glycoprotein/MDR inhibitor), ZD-9331, ZD-1839 (IRESSA®), andZoledronate (Zometa®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, prevention, amelioration and/orcure of cancers and premalignant conditions.

In further specific embodiments, polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof may beadministered in combination with one or more combinations of therapeuticagents useful in the treatment, prevention, amelioration and/or cure ofcancers and premalignant conditions including, but not limited to,9-aminocamptothecin+G-CSF, Adriamycin®+Blenoxane+Vinblastine+Dacarbazine(ABVD), BCNU (Carmustine)+Etoposide+Ara-C (Cytarabine)+Melphalen (BEAM),Bevacizumab®+Leucovorin, Bleomycin+Etoposide+Platinol® (Cisplatin)(BEP),Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+Vincristine+Procarbazine+Prednisone(BEACOPP), Bryostatin+Vincristine, Busulfan+Melphalan,Carboplatin+Cereport®, Carboplatin+Cyclophosphamide,Carboplatin+Paclitaxel, Carboplatin+Etoposide+Bleomycin (CEB),Carboplatin+Etoposide+Thiotepa, Cisplatin+Cyclophosphamide,Cisplatin+Docetaxel, Cisplatin+Doxorubicin, Cisplatin+Etoposide,Cisplatin+Gemcitabine, Cisplatin+Interferon alpha, Cisplatin+Irinotecan,Cisplatin+Paclitaxel, Cisplatin+Teniposide, Cisplatin+Vinblastine,Cisplatin+Vindesine, Cisplatin+Vinorelbine,Cisplatin+Cytarabine+Ifosfamide, Cisplatin+Ifosfamide+Vinblastine,Cisplatin+Vinblastine+Mitomycin C, Cisplatin+Vincristine+Fluorouracil,Cisplatin+Vincristine+Lomustine, Cisplatin+Vinorelbine+Gemcitabine,Cisplatin+Carmustine+Dacarbazine+Tamoxifen,Cisplatin+Cyclophosphamide+Etoposide+Vincristine, Cisplatin(Platinol®)+Oncovin®+Doxorubicin (Adrianycin®)+Etoposide (CODE),Cisplatin+Cytarabine+Ifosfamide+Etoposide+Methotrexate,Cyclophosphamide+Adriamycin® (Doxorubicin), Cyclophosphamide+Melphalan,Cyclophosphamide+SCH 6636, Cyclophosphamide+Adriamycin®+Cisplatin(Platinol®) (CAP), Cyclophosphamide+Adriamycin®+Vincristine (CAV),Cyclophosphamide+Doxorubicin+Teniposide+Prednisone,Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon alpha,Cyclophosphamide+Epirubicin+Cisplatin (Platinol®) (CEP),Cyclophosphamide+Epirubicin+Fluorouracil,Cyclophosphamide+Methotrexate+Fluoruracil (CMF),Cyclophosphamide+Methotrexate+Vincristine (CMV),Cyclophosphamide+Adriamycing+Methotrexate+Fluorouracil (CAMF),Cyclophosphamide+Adriamycin®+Methotrexate+Procarbazine (CAMP),Cyclophosphamide+Adriamycin®+Vincristine+Etoposide (CAV-E),Cyclophosphamide+Adriamycing+Vincristine+Prednisone (CHOP),Cyclophosphamide+Novantrone® (Mitoxantrone)+Vincristine(Oncovorin)+Prednisone (CNOP),Cyclophosphamide+Adriamycin®+Vincristine+Prednisone+Rituximab(CHOP+Rituximab), Cyclophosphamide+Adriamycin®+Vincristine+Teniposide(CAV-T), Cyclophosphamide+Adriamycin®+Vincristine alternating withPlatinol®+Etoposide (CAV/PE), Cyclophosphamide+BCNU (Carmustine)+VP-16(Etoposide) (CBV), Cyclophosphamide+Vincristine+Prednisone (CVP),Cyclophosphamide+Oncovin®+Methotrexate+Fluorouracil (COMF),Cytarabine+Methotrexate, Cytarabine+Bleomycin+Vincristine+Methotrexate(CytaBOM), Dactinomycin+Vincristine, Dexamethasone+Cytarabine+Cisplatin(DHAP), Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE),Docetaxel+Gemcitabine, Docetaxel+Vinorelbine,Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+Prednisone(Stanford V), Epirubicin+Gemcitabine, Estramustine+Docetaxel,Estramustine+Navelbine, Estramustine+Paclitaxel,Estramustine+Vinblastine, Etoposide (Vepesid®)+Ifosfamide+Cisplatin(Platinol®) (VIP), Etoposide+Vinblastine+Adriamycin (EVA), Etoposide(Vepesid®)+Ifosfamide+Cisplatin+Epirubicin (VIC-E),Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP),Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC),Fludarabine+Mitoxantrone+Dexamethasone (FMD),Fludarabine+Dexamethasone+Cytarabine (ara-C)+Cisplatin (Platinol®)(FluDAP), Fluorouracil+Bevacizumab®, Fluorouracil+CeaVac®,Fluorouracil+Leucovorin, Fluorouracil+Levamnisole,Fluorouracil+Oxaliplatin, Fluorouracil+Raltitrexed, Fluorouracil+SCH6636, Fluorouracil+Trimetrexate, Fluorouracil+Leucovorin+Bevacizumab®,Fluorouracil+Leucovorin+Oxaliplatin,Fluorouracil+Leucovorin+Trimetrexate, Fluorouracil+Oncovin®+Mitomycin C(FOMi), Hydrazine+Adriamycin®+Methotrexate (HAM), Ifosfamide+Docetaxel,Ifosfamide+Etoposide, Ifosfamide+Gemcitabine, Ifosfamide+Paclitaxel,Ifosfamide+Vinorelbine, Ifosfamide+Carboplatin+Etoposide (ICE),Ifosfamide+Cisplatin+Doxorubicin, Irinotecan+C225 (Cetuximab®),Irinotecan+Docetaxel, Irinotecan+Etoposide, Irinotecan+Fluorouracil,Irinotecan+Gemcitabine, Mechlorethamine+Oncovin®(Vincristine)+Procarbazine (MOP), Mechlorethamine+Oncovin®(Vincristine)+Procarbazine+Prednisone (MOPP),Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate+Interferonalpha, Methotrexate+Vinblastine, Methotrexate+Cisplatin, Methotrexatewith leucovorinrescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone(m-BACOD), Mitomycin C+Ifosfamide+Cisplatin (Platinol®) (MIP), MitomycinC+Vinblastine+Paraplatin® (MVP), Mitoxantrone+Hydrocortisone,Mitoxantrone+Prednisone, Oncovin®+SCH 6636, Oxaliplatin+Leucovorin,Paclitaxel+Doxorubicin, Paclitaxel+SCH 6636, Paraplatin®+Docetaxel,Paraplatin®V+Etoposide, Paraplatin®+Gemcitabine, Paraplatin®+Interferonalpha, Paraplatin®+Irinotecan, Paraplatin®+Paclitaxel,Paraplatin®)+Vinblastine, Carboplatin (Paraplatin®)+Vincristine,Paraplatin®+Vindesine, Paraplatin®+Vinorelbine, Pemetrexeddisodium+Gemcitabine, Platinol® (Cisplatin)+Vinblastine+Bleomycin (PVB),Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide (ProMACE),Procarbazine+Lomustine, Procarbazine+Lomustine+Vincristine,Procarbazine+Lomustine+Vincristine+Thioguanine,Procarbazine+Oncovin®+CCNU®+Cyclophosphamide (POCC),Quinine+Doxorubicin, Quinine+Mitoxantrone+Cytarabine,Thiotepa+Etoposide, Thiotepa+Busulfan+Cyclophosphamide,Thiotepa+Busulfan+Melphalan, Thiotepa+Etoposide+Carmustine,Thiotepa+Etoposide+Carboplatin, Topotecan+Paclitaxel,Trimetrexate+Leucovorin, Vinblastine+Doxorubicin+Thiotepa,Vinblastine+Bleomycin+Etoposide+Carboplatin,Vincristine+Lomustine+Prednisone, Vincristine(Oncovin®)+Adriamycin®+Dexamethasone (VAD), Vincristine(Oncovin®)+Adriamycing+Procarbazine (VAP),Vincristine+Dactinomycin+Cyclophosphamide, and Vinorelbine+Gemcitabine.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedcombinations of therapeutic agents in the treatment, prevention,amelioration and/or cure of cancers and premalignant conditions.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents described above to treat, prevent, ameliorateand/or cure cancers and premalignant conditions of any tissue known toexpress DR5 receptor.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more therapeutic agentsdescribed above to treat, prevent, ameliorate and/or cure cancers andpremalignant conditions of any tissue known to express DR5 receptor.

Tissues known to express DR5 receptor include, but are not limited to,heart, placenta, lung, liver, skeletal muscle, pancreas, spleen, thymus,prostate, testis, uterus, ovary, small intestine, colon, brain kidney,bone marrow, skin, pituitary, cartilage and blood.

In specific embodiments polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof may be administeredin combination with one or more therapeutic agents, as described above,in the treatment, prevention, amelioration and/or cure of solid tissuecancers (e.g., skin cancer, prostate cancer, pancreatic cancer, hepaticcancer, lung cancer, ovarian cancer, colorectal cancer, head and necktumors, breast tumors, endothelioma, osteoblastoma, osteoclastoma,Ewing's sarcoma, and Kaposi's sarcoma), as well as hematological cancers(e.g., leukemia, acute lymphocytic leukemia, chronic lymphocyticleukemia, non-Hodgkin's lymphoma, multiple myeloma).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more therapeutic agents, asdescribed above, in the treatment, prevention, amelioration and/or cureof solid tissue cancers (e.g., skin cancer, prostate cancer, pancreaticcancer, hepatic cancer, lung cancer, ovarian cancer, colorectal cancer,head and neck tumors, breast tumors, endothelioma, osteoblastoma,osteoclastoma, Ewing's sarcoma, and Kaposi's sarcoma), as well ashematological cancers (e.g., leukemia, acute lymphocytic leukemia,chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma).

In specific embodiments polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof may be used totreat, ameliorate and/or prevent skin cancers including, but not limitedto, basal cell carcinoma, squamous cell carcinoma and malignantmelanoma. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent skin cancers.

In preferred embodiments agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent skin cancersincluding, but not limited to, basal cell carcinoma, squamous cellcarcinoma and malignant melanoma. Agonists and/or antagonists of thepresent invention may be used in combination with one or more surgicaland/or radiological procedures and/or therapeutic agents to treat,ameliorate and/or prevent skin cancers.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of skin cancers andpremalignant conditions including, but not limited to, Bleomycin(Blenoxane®), Cannustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®),Cisplatin (Platinol®, CDDP), Dacarbazine (DTIC), Interferon alpha 2b(Intron A®), Interleukin-2 (ProleiukinR®), Tamoxifen (Nolvadex®,Temozolamide (Temodar®, NSC 362856), Vinblastine (Velban®, VLB),Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®), and Vindesine(Eldisine®, Fildesin®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofskin cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofskin cancers and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Cisplatin+Carmustine+Dacarbazine+Tamoxifen.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of skin cancers and premalignant conditions.

In further specific embodiments, polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent head and neck cancers including braincancers. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent head and neck cancersincluding brain cancers. Brain cancers which may be treated usingpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to, gliomas suchas astrocytomas and oligodendromas, non-glial tumors such as neuronal,meningeal, ependymal and choroid plexus cell tumors, and metastaticbrain tumors such as those originating as breast, lung, prostate andskin cancers.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent head and neckcancers including brain cancers. Agonists and/or antagonists of thepresent invention may be used in combination with one or more surgicaland/or radiological procedures and/or therapeutic agents to treat,ameliorate and/or prevent head and neck cancers including brain cancers.Brain cancers which may be treated using agonists and/or antagonists ofthe present invention include, but are not limited to, gliomas such asastrocytomas and oligodendromas, non-glial tumors such as neuronal,meningeal, ependymal and choroid plexus cell tumors, and metastaticbrain tumors such as those originating as breast, lung, prostate andskin cancers.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat brain tumors. In one preferred embodiment, agonists ofthe invention are used to treat glioblastoma multiforme.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more radiological procedures useful in the treatment of brain cancersincluding, but not limited to, external beam radiation therapy,stereotactic radiation therapy, conformal radiation therapy,intensity-modulated radiation therapy (IMRT), and radiosurgery.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more radiological proceduresuseful in the treatment of brain cancers including, but not limited to,external beam radiation therapy, stereotactic radiation therapy,conformal radiation therapy, intensity-modulated radiation therapy(IMRT), and radiosurgery.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of brain cancers andpremalignant conditions, including, but not limited to, Bleomycin(Blenoxane®), Busulfan (Busulfex®, Myleran®), Carboplatin (Paraplatin®,CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Cisplatin(Platinol®, CDDP), Cisplatin-epinephrine gel (IntraDose®, FocaCist®),Cyclophosphamide (Cytoxan®, CTX), Cytarabine (Cytosar-U®, ara-C,cytosine arabinoside, DepoCyt®), Dacarbazine (DTIC®), Dactinomycin(Cosmegen®), Daunorubicin (Daunomycin, DaunoXome®, Daunorubicin®,Cerubidine®), Docetaxel (Taxotere®, Taxane®), Dexamethasone (Decadron®),Etoposide phosphate (Etopophos®), Etoposide (VP-16, Vepesid®),Fluorouracil (5-FU, Adrucil®), Hydroxyurea (Hydrea®), Ifosfamide(IFEX®), Lomustine (CCNU®, CeeNU®), Melphalan (L-PAM, Alkeran®,Phenylalanine mustard), Mercaptopurine (6-mercaptopurine, 6-MP),Methchlorethamine (Nitrogen Mustard, HN₂, Mustargen®),Methotrexate®(MTX, Mexate®, Folex®), Paclitaxel (Paxene®, Taxol®),Paclitaxel-DHA (Taxoprexin®), Procarbazine (Matulane®), Temozolamide(Temodar®, NSC 362856), Teniposide (VM-26, Vumon®), Thioguanine(6-thioguanine, 6-TG), Thiotepa (triethylenethiophosphaoramide),Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®), andVincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbrain cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment of braincancers and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, 81C6 (Anti-tenascin monoclonal antibody), BIBX-1382, Cereport®(Lobradimil®, RMP-7), Cilengitide® (EMD-121974, integrin alphavbeta3antagonist), CMT-3 (Metastat®), Cotara® (chTNT-1/B, [¹³¹I]-chTNT-1/B),CP IL-4-toxin (IL-4 fusion toxin), Fenretinide® (4HPR), Fotemustine(Muphoran®, Mustophoran®), Gemcitabine (Gemto®, Gemzar®), Hypericin®(VIMRxyn®), Imatinib mesylate (STI-571, Imatinib®, Glivec®, Gleevec®,Ab1 tyrosine kinase inhibitor), Irinotecan (Camptosar®, CPT-11,Topotecin®, CaptoCPT-1), Leflunomide (SU-101, SU-0200), Mivobulinisethionate (CI-980), 06-benzylguanine (BG, Procept®), Prinomastat®(AG-3340, MMP inhibitor), R115777 (Zarnestra®), SU6668 (PDGF-TKinhibitor), T-67 (T-138067, T-607), Tamoxifen (Nolvadex®), Tf-CRM107(Transferrin-CRM-107), Thalidomide and thalidomide analogs, includingbut not limited to, lenalidomide (CC-5013, REVLIMID®) and CC-4047(ACTIMID™), Tiazofurin (Thiazole®), Vapreotide® (BMY-41606), Vinorelbine(Navelbine®), and XR-5000 (DACA).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbrain cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofbrain cancers and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Busulfan+Melphalan, Carboplatin+Cereport®, Carboplatin+Etoposide,Carboplatin+Etoposide+Thiotepa, Cisplatin+Etoposide,Cisplatin+Cytarabine+Ifosfamide, Cisplatin+Vincristine+Lomustine,Cisplatin+Cyclophosphamide+Etoposide+Vincristine,Cisplatin+Cytarabine+Ifosfamide+Etoposide+Methotrexate,Cyclophosphamide+Melphalan, Cytarabine+Methotrexate,Dactinomycin+Vincristine, Mechlorethamine+Oncovin®(Vincristine)+Procarbazine (MOP), Mechlorethamine+Oncovin®(Vincristine)+Procarbazine+Prednisone (MOPP), Carboplatin(Paraplatin®)+Etoposide, Carboplatin (Paraplatin®)+Vincristine,Procarbazine+Lomustine, Procarbazine+Lomustine+Vincristine,Procarbazine+Lomustine+Vincristine+Thioguanine, Thiotepa+Etoposide,Thiotepa+Etoposide+Carmustine, Thiotepa+Etoposide+Carboplatin,Vinblastine+Bleomycin+Etoposide+Carboplatin, andVincristine+Lomustine+Prednisone.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedcombinations of therapeutic agents in the treatment, amelioration and/orprevention of brain cancers and premalignant conditions.

In further particular embodiments, polynucleotides and/or polypeptidesof the invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent breast cancer and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent breast cancer. Breast cancerswhich may be treated using polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof include, but arenot limited to, ductal carcinoma, stage 1, stage 11, stage III and stage1V breast cancers as well as invasive breast cancer and metastaticbreast cancer.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent breast cancer andpremalignant conditions. Agonists and/or antagonists of the presentinvention may be used, in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent breast cancer and premalignant conditions. Breast cancerswhich may be treated using agonists and/or antagonists of the presentinvention include, but are not limited to, ductal carcinoma, stage I,stage 11, stage III and stage 1V breast cancers as well as invasivebreast cancer and metastatic breast cancer.

In preferred embodiment, agonists and/or antagonists of the inventionare used to treat metastatic breast cancer.

In other preferred embodiments, agonists and/or antagonists of thepresent invention are administered in combination with one or moresurgical and/or radiological procedures useful in the treatment ofbreast cancer and premalignant conditions.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of breast cancer andpremalignant conditions, including, but not limited to, Amifostine(Ethyol®), Aminoglutethimide (Cytadren®), Anastrozole (Arimidex®),Bleomycin (Blenoxane®), Capecitabine (Xeloda®, Doxifluridine®, oral5-FU), Cisplatin (Platinol®, CDDP), Cisplatin-epinephrine gel(IntraDose®, FocaCist®), Cyclophosphamide (Cytoxan®, Neosar®, CTX),Docetaxel (Taxotere®, Taxane®), Doxorubicin (Adriamycin®, Doxil®,Rubex®), Epirubicin (Ellence®, EPI, 4′ epi-doxorubicin), Exemestane(Aromasin®, Nikidess®R), Fadrozole (Afema®, Fadrozole hydrochloride,Arensin®), Fluorouracil (5-FU, Adrucil®, Fluoroplex®, Efudex®),Herceptin® (Trastuzumab®, Anti-HER-2 monoclonal antibody, Anti-EGFR-2MAb), Ifosfamide (IFEX®), Letrozole (Femara®), Leucovorin (Leucovorin®,Wellcovorin®), Mechlorethamine (Nitrogen Mustard, HN₂, Mustargen®),Megestrol acetate (Megace®, Pallace®), Melphalan (L-PAM, Alkeran®,Phenylalanine mustard), Methotrexate® (MTX, Mexate®, Folex®),Methyltestosterone (Android-10®, Testred®, Virilon®), Mitomycin C(Mitomycin®, Mutamycin®, Mito Extra®), Orzel®(Tegafur+Uracil+Leucovorin), Paclitaxel (Paxene®, Taxol®), Sobuzoxane(MST-16, Perazolin®), Tamoxifen (Nolvadex®, Testosterone (Andro®,Androderm®, Testoderm TTS®, Testoderm®, Depo-Testosterone®, Androgel®,depoAndro®), Vinblastine (Velban®, VLB), Vincristine (Oncovin®, OncoTCS®, VCR, Leurocristine®), and Vinorelbine (Navelbine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbreast cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment of breastcancer and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Aldesleukin (IL-2, Proleukin®), Altretamine (Hexylen®,hexamethylmelamine, Hexastat®), Angiostatin, Annamycin (AR-522,annamycin LF, Aronex®)), Biricodar dicitrate (Incel®, Incel MDRInhibitor), Boronated Protoporphyrin Compound (PDIT, PhotodynamicImmunotherapy), Bryostatin-1 (Bryostatin, BMY-45618, NSC-339555),Busulfan (Busulfex®, Myleran®), Carmustine (DTI-015, BCNU, BiCNU,Gliadel Wafer®), D-limonene, Dacarbazine (DTIC), Daunorubicin(Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®), Dolastatin-10(DOLA-10, NSC-376128), DPPE, DX-8951f (DX-8951), EMD-121974, Endostatin,EO9 (EO1, EO4, EO68, EO70, EO72), Etoposide phosphate (Etopophos®),Etoposide (VP-16, Vepesid®), Fluasterone, Fludarabine (Fludara®V, FAMP),Flutamide (Eulexin®), Formestane (Lentaron®), Fulvestrant (Faslodex®),Galarubicin hydrochloride (DA-125), Gemcitabine (Gemto®, Gemzar®),Her-2/Neu vaccine, Hydroxyurea (Hydrea®), Idarubicin (Idamycin®, DMDR,IDA), Interferon alpha 2a (Intron A®), Interferon gamma(Gamma-interferon, Gamma 100®, Gamma-IF), Irinotecan (Camptosar®,CPT-11, Topotecin®, CaptoCPT-1), Ketoconazole (Nizoral®), KRN-8602 (MX,MY-5, NSC-619003, MX-2), L-asparaginase (Elspar®), Leuprolide acetate(Viadur®, Lupron®), Lomustine (CCNU®, CeeNU®), LY-335979, Mannan-MUC1vaccine, 2-Methoxyestradiol (2-ME, 2-ME2), Mitoxantrone (Novantrone®,DHAD), Motexafin Lutetium (Lutrin®, Optrin®, Lu-Tex®, lutetiumtexaphyrin, Lucyn®, Antrin®), MPV-2213ad (Finrozole®), MS-209, Muc-1vaccine, NaPro Paclitaxel, Perillyl alcohol (perilla alcohol, perillicalcohol, perillol, NSC-641066), Pirarubicin (THP), Procarbazine(Matulane®), Providence Portland Medical Center Breast Cancer Vaccine,Pyrazoloacridine (NSC-366140, PD-115934), Raloxifene hydrochloride(Evista®, Keoxifene hydrochloride), Raltitrexed (Tomudex®, ZD-1694),Rebeccamycin, Streptozocin (Zanosar®), Temozolamide (Temodar®, NSC362856), Theratope, Thiotepa (triethylenethiophosphaoramide, Thioplex®),Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®), Toremifene(Estrimex®, Fareston®), Trilostane (Modrefen®), and XR-9576 (XR-9351,P-glycoprotein/MDR inhibitor).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofbreast cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofbreast cancer which may be administered in combination withpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to,Cyclophosphamide+Adriamycin® (Doxorubicin),Cyclophosphamide+Epirubicin+Fluorouracil,Cyclophosphamide+Methotrexate+Fluorouracil (CMF),Paclitaxel+Doxorubicin, and Vinblastine+Doxorubicin+Thiotepa.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of breast cancers and premalignant conditions.

In further specific embodiments, polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent lung cancer and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent lung cancer. Lung cancerwhich may be treated using polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof includes, but isnot limited to, non-small cell lung cancer (NSCLC) including early stageNSCLC (i.e., Stage IA/IB and Stage IIA/IIB), Stage IIIA NSCLC, Stage IIA(unresectable)/IIIB NSCLC and Stage IV NSCLC, small cell lung cancer(SCLC) including limited stage SCLC and extensive stage SCLC as well asMalignant Pleural Mesothelioma.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent lung cancer andpremalignant conditions. Agonists and/or antagonists of the presentinvention may be used in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent lung cancer and premalignant conditions. Lung cancerwhich may be treated using agonists and/or antagonists of the presentinvention includes, but is not limited to, non-small cell lung cancer(NSCLC) including early stage NSCLC (i.e., Stage IA/IB and StageIIA/IIB), Stage IIIA NSCLC, Stage IIA (unresectable)/IIIB NSCLC andStage IV NSCLC, small cell lung cancer (SCLC) including limited stageSCLC and extensive stage SCLC as well as Malignant Pleural Mesothelioma.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat non-small cell lung cancers.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of lung cancer andpremalignant conditions, including, but not limited to, BAY 43-9006 (Rafkinase inhibitor), Carboplatin (Paraplatin®, CBDCA), Chlorambucil(Leukeran®), Cisplatin (Platinol®, CDDP), Cisplatin-epinephrine gel(IntraDose®, FocaCist®), Cyclophosphamide (Cytoxan®, Neosar®, CTX),Docetaxel (Taxotere®, Taxane®), Doxorubicin (Adriamycin®, Doxil®,Rubex®), Edatrexate, Epirubicin (Ellence®, EPI, 4′ epi-doxorubicin),Etoposide phosphate (Etopophos®), Etoposide (VP-16, Vepesid®),Gemcitabine (Gemto®, Gemzar®), Herceptin® (Trastuzumab®, Anti-HER-2monoclonal antibody, Anti-EGFR-2 MAb), Ifosfamide (IFEX®), Irinotecan(Camptosar®, CPT-11, Topotecin®, CaptoCPT-1), Lomustine (CCNU®, CeeNU®),Mechlorethamine (Nitrogen Mustard, HN₂, Mustargen®), Melphalan (L-PAM,Alkeran®, Phenylalanine mustard), Methotrexate® (MTX, Mexate®, Folex®),Mitomycin C (Mitomycin®, Mutamycin®, Mito Extra®), Paclitaxel (Paxene®,Taxol®), Paclitaxel-DHA (Taxoprexin®), Porfimer sodium (Photofrin®),Procarbazine (Matulane®), SKI-2053R(NSC-D644591), Teniposide (VM-26,Vumon®), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®),Vinblastine (Velban®, VLB), Vincristine (Oncovin®, Onco TCS®, VCR,Leurocristine®), Vindesine (Eldisine®, Fildesin®), and Vinorelbine(Navelbine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention oflung cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment of lungcancer and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, ABX-EGF (anti-EGFr MAb), Acetyldinaline (CI-994), AG-2034 (AG-2024,AG-2032, GARFT [glycinamide ribonucleoside transformylase] inhibitor),Alanosine, Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071),Angiostatin, Aplidine (Aplidin®, Aplidina®), BBR 3464, Bexarotene(Targretin®, LGD1069), BIBH-1 (Anti-FAP MAb), BIBX-1382, BLP-25 (MUC-1peptide), Bryostatin-1 (Bryostatin®V, BMY-45618, NSC-339555), Budesonide(Rhinocort®), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb, Cetuximab®),Capecitabine (Xeloda®, Doxifluridine®, oral 5-FU), Carboxyamidotriazole(NSC 609974, CAI, L-651582), CEA-cide®) (Labetuzumab®, Anti-CEAmonoclonal antibody, hMN-14), Cereport® (Lobradimil®, RMP-7), CI-1033(Pan-erbB RTK inhibitor), Cilengitide® (EMD-121974, integrin alphavbeta3antagonist), 9-cis retinoic acid (9-cRA), Cisplatin-liposomal (SPI-077),CMB-401 (Anti-PEM MAb/calicheamycin), CMT-3 (Metastat®), CP-358774(Tarceva®, OSI-774, EGFR inhibitor), CT-2584 (Apra®), DAB389-EGF (EGFfusion toxin), DeaVac® (CEA anti-idiotype vaccine), Decitabine(5-aza-2′-deoxyytidine), Diethylnorspermine (DENSPM),Dihydro-5-azacytidine, EGF-P64k Vaccine, Endostatin, Etanidazole(Radinyl®), Exetecan mesylate (DX-8951, DX-8951f), Exisulind (SAAND,Aptosyn®, cGMP-PDE2 and 5 inhibitor), FK-317 (FR-157471, FR-70496),Flavopiridol (HMR-1275), Fotemustine (Muphoran®, Mustophoran®), G3139(Genasense®, GentaAnticode®, Bcl-2 antisense), Gadolinium texaphyrin(Motexafin gadolinium, Gd-Tex®, Xcytrin®), GBC-590, GL331, Galarubicinhydrochloride (DA-125), Glufosfamide® (O-D-glucosyl-isofosfamidemustard, D19575, INN), GVAX (GM-CSF gene therapy), INGN-101 (p53 genetherapy/retrovirus), INGN-201 (p53 gene therapy/adenovirus), Irofulven(MGI-114), ISIS-2053, ISIS-3521 (PKC-alpha antisense), ISIS-5132(K-ras/raf antisense), Isotretinoin (13-CRA, 13-cis retinoic acid,Accutane®), Lometrexol (T-64, T-904064), Marimastat® (BB-2516, TA-2516,MMP inhibitor), MDX-447 (BAB-447, EMD-82633, H-447,anti-EGFr/FcGammaR1r), MGV, Mitumomab® (BEC-2, EMD-60205), Mivobulinisethionate (CI-980), Neovastat® (AE-941, MMP inhibitor), NPI-0052(proteasome inhibitor), Onconase (Ranpirnase®), Onyx-015 (p53 genetherapy), Pemetrexed disodium (Alimta®, MTA, multitargeted antifolate,LY 231514), Pivaloyloxymethyl butyrate (AN-9, Pivanex®), Prinomastat®(AG-3340, MMP inhibitor), PS-341 (LDP-341, 26S proteasome inhibitor),Pyrazoloacridine (NSC-366140, PD-115934), R115777 (Zamestra®),Raltitrexed (Tomudex®, ZD-1694), R-flurbiprofen (Flurizan®, E-7869,MPC-7869), RFS-2000 (9-nitrocamptothecan, 9-NC, Rubitecan®), RSR-13(GSJ-61), Satraplatin (BMS-182751, JM-216), SCH-66336, Sizofilan® (SPG,Sizofuran®, Schizophyllan®, Sonifilan®), Squalamine (MSI-1256F),SR-49059 (vasopressin receptor inhibitor, Vla), SU5416 (Semaxanib®),VEGF inhibitor), Taurolidine (Taurolin®), Temozolamide (Temodar®, NSC362856), Thalidomide and thalidomide analogs, including but not limitedto, lenalidomide (CC-5013, REVLIMID®) and CC-4047 (ACTIMID™), Thymosinalpha I (Zadaxin®, Thymalfasin®), Tirapazamine (SR-259075, SR-4233,Tirazone®, Win-59075), TNP-470 (AGM-1470), TriAb® (anti-idiotypeantibody immune stimulator), Tretinoin (Retin-A®, Atragen®, ATRA,Vesanoid®), Troxacitabine (BCH-204, BCH-4556, Troxatyl®D), Vitaxin®D(LM-609, integrin alphavbeta3 antagonistic MAb), XR-9576(P-glycoprotein/MDR inhibitor), and ZD-1839 (IRESSA®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention oflung cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment oflung cancer and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Cisplatin+Docetaxel, Cisplatin+Etoposide, Cisplatin+Gemcitabine,Cisplatin+Interferon alpha, Cisplatin+Irinotecan, Cisplatin+Paclitaxel,Cisplatin+Teniposide, Cisplatin+Vinblastine, Cisplatin+Vindesine,Cisplatin+Vinorelbine, Cisplatin+Vinblastine+Mitomycin C,Cisplatin+Vinorelbine+Gemcitabine, Cisplatin(Platinol®)+Oncovin®+Doxorubicin (Adriamycin®)+Etoposide (CODE),Cyclophosphamide+Adriamycing+Cisplatin (Platinol®) (CAP),Cyclophosphamide+Adriamycing+Vincristine (CAV),Cyclophosphamide+Epirubicin+Cisplatin (Platinol®) (CEP),Cyclophosphamide+Methotrexate+Vincristine (CMV),Cyclophosphamide+Adriamycin®, Methotrexate+Fluorouracil (CAMF),Cyclophosphamide+Adriamycin®, Methotrexate+Procarbazine (CAMP),Cyclophosphamide+Adriamycin®, Vincristine+Etoposide (CAV-E),Cyclophosphamide+Adriamycin®, Vincristine+Teniposide (CAV-T),Cyclophosphamide+Oncovin®, Methotrexate+Fluorouracil (COMF),Cyclophosphamide+Adriamycin®+Vincristine, alternating withCisplatin+Etoposide (CAV/PE), Docetaxel+Gemcitabine,Docetaxel+Vinorelbine, Etoposide (Vepesid®)+Ifosfamide+Cisplatin(Platinol®) (VIP), Etoposide (Vepesid®)+Ifosfamide, Cisplatin+Epirubicin(VIC-E), Fluorouracil+Oncovin®+Mitomycin C (FOMi),Hydrazine+Adriamycin®+Methotrexate (HAM), Ifosfamide+Docetaxel,Ifosfamide+Etoposide, Ifosfamide+Gemcitabine, Ifosfamide+Paclitaxel,Ifosfamide+Vinorelbine, Ifosfamide+Carboplatin+Etoposide (ICE),Irinotecan+Docetaxel, Irinotecan+Etoposide, Irinotecan+Gemcitabine,Methotrexate+Cisplatin, Methotrexate+Interferon alpha,Methotrexate+Vinblastine, Mitomycin C+Ifosfamide+Cisplatin (Platinol®)(MIP), Mitomycin C+Vinblastine+Paraplatin® (MVP), Paraplatin®+Docetaxel,Paraplatin®+Etoposide, Paraplatin®+Gemcitabine, Paraplatin®+Interferonalpha, Paraplatin®+Irinotecan, Paraplatin®+Paclitaxel,Paraplatin®)+Vinblastine, Paraplatin®+Vindesine,Paraplatin®+Vinorelbine, Procarbazine+Oncovin®+CCNU®(Lomustine)+Cyclophosphamide (POCC), Vincristine(Oncovin®)+Adriamycin®+Procarbazine (VAP), and Vinorelbine+Gemcitabine.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of lung cancers and premalignant conditions.

In further particular embodiments, polynucleotides and/or polypeptidesof the invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent colorectal cancer and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent colorectal cancer andpremalignant conditions. Colorectal cancers which may be treated usingpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to, colon cancer(e.g., early stage colon cancer (stage I and II), lymph node positivecolon cancer (stage III), metastatic colon cancer (stage 1V)) and rectalcancer.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent colorectal cancerand premalignant conditions. Agonists and/or antagonists of the presentinvention may be used in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent colorectal cancer and premalignant conditions. Colorectalcancers which may be treated using agonists and/or antagonists of thepresent invention include, but are not limited to, colon cancer (e.g.,early stage colon cancer (stage I and II), lymph node positive coloncancer (stage III), metastatic colon cancer (stage 1V)) and rectalcancer.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat colon cancer and premalignant conditions.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of colorectal cancerand premalignant conditions, including, but not limited to, Capecitabine(Xeloda®, Doxifluridine®, oral 5-FU), Fluorouracil (5-FU, Adrucil®,Fluoroplex®, Efudex®), Irinotecan (Camptosar®, CPT-11, Topotecin®,CaptoCPT-1), Leucovorin (Leucovorin®, Welicovorin®), and Levamisole(Ergamisol®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofcolorectal cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofcolorectal cancer and premalignant conditions which may be administeredin combination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Fluorouracil+Leucovorin and Fluorouracil+Levamisole.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of colorectal cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment ofcolorectal cancer and premalignant conditions which may be administeredin combination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aplidine(Aplidin®, Aplidina®), Bevacizumab® (Anti-VEGF monoclonal antibody,rhuMAb-VEGF), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb, Cetuximab®),C242-DM1 (huC242-DM1), CC49-zeta gene therapy, CEA-cide® (Labetuzumab®,Anti-CEA monoclonal antibody, hMN-14), CeaVac® (MAb 3H1), CP-609754,CTP-37 (Avicine®, hCG blocking vaccine), Declopramide (Oxi-104),Eniluracil (776c85), F19 (Anti-FAP monoclonal antibody, iodinatedanti-FAP MAb), FMdC (KW-2331, MDL-101731), FUDR (Floxuridine®),Gemcitabine (Gemto®, Gemzar®), Herceptin®(Trastuzumab®, Anti-HER-2monoclonal antibody, Anti-EGFR-2 MAb), Intoplicine (RP 60475), L-778123(Ras inhibitors), Leuvectin® (cytofectin+IL-2 gene, IL-2 gene therapy),MN-14 (Anti-CEA immunoradiotherapy, ¹³¹I-MN-14, ¹⁸⁸Re-MN-14),OncoVAX-CL, OncoVAX-CL-Jenner (GA-733-2 vaccine) Orzel®(Tegafur+Uracil+Leucovorin), Oxaliplatin (Eloxatine®, Eloxatin®),Paclitaxel-DHA (Taxoprexin®), Pemetrexed disodium (Alimta®, MTA,multitargeted antifolate, LY 231514), R115777 (Zarnestra®), Raltitrexed(Tomudex®, ZD-1694), SCH 66336, SU5416 (Semaxanib®, VEGF inhibitor),Tocladesine (8-Cl-cAMP), Trimetrexate (Neutrexin®), TS-1, and ZD-9331.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofcolorectal cancers and premalignant conditions.

Further exemplary combinations of therapeutic agents useful in thetreatment of colorectal cancer and premalignant conditions which may beadministered in combination with polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof include, butare not limited to, Aminocamptothecin+G-CSF, Bevacizumab®+Fluorouracil,Bevacizumab®+Leucovorin, Bevacizumab®+Fluorouracil+Leucovorin,Cyclophosphamide+SCH 6636, Fluorouracil+CeaVac®,Fluorouracil+Oxaliplatin, Fluorouracil+Raltitrexed, Fluorouracil+SCH6636, Fluorouracil+Trimetrexate, Fluorouracil+Leucovorin+Oxaliplatin,Fluorouracil+Leucovorin+Trimetrexate, Irinotecan+C225 (Cetuximab®),Oncovin®+SCH 6636, Oxaliplatin+Leucovorin, Paclitaxel+SCH 6636,Pemetrexed disodium+Gemcitabine, and Trimetrexate+Leucovorin.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of colorectal cancers and premalignant conditions.

In further specific embodiments, polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent prostate cancer and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent prostate cancer andpremalignant conditions. Prostate cancer which may be treated usingpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof includes, but is not limited to, benignprostatic hyperplasia, malignant prostate cancer (e.g., stage 1, stage11, stage III or stage 1V) and metastatic prostate cancer.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent prostate cancerand premalignant conditions. Agonists and/or antagonists of the presentinvention may be used in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent prostate cancer and premalignant conditions. Prostatecancer which may be treated using polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof includes, butis not limited to, benign prostatic hyperplasia, malignant prostatecancer (e.g., stage 1, stage 11, stage III or stage 1V) and metastaticprostate cancer.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat malignant prostate cancer. In other preferredembodiments, agonists and/or antagonists of the invention are used totreat metastatic prostate cancer.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more surgical, radiological and/or hormonal procedures useful in thetreatment of prostate cancer and premalignant conditions including, butnot limited to, prostatectomy (e.g., radical retropubic prostatectomy),external beam radiation therapy, brachytherapy, orchiectomy and hormonetreatment (e.g., LHRH agonists, androgen receptor inhibitors).

In preferred embodiments, agonists and/or antagonists of the presentinvention may be administered in combination with one or more surgical,radiological and/or hormonal procedures useful in the treatment ofprostate cancer and premalignant conditions including, but not limitedto, prostatectomy (e.g., radical retropubic prostatectomy), externalbeam radiation therapy, brachytherapy, orchiectomy and hormone treatment(e.g., LHRH agonists, androgen receptor inhibitors).

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of prostate cancerand premalignant conditions including, but not limited to,Aminoglutethimide (Cytadren®), Biclutamide (Casodex®), Cyclophosphamide(Cytoxan®g, Neosar®, CTX), Diethylstilbestrol (DES), Doxorubicin(Adriamycin®, Doxil®, Rubex®), Flutamide (Eulexin®), Hydrocortisone,Ketoconazole (Nizoral®), Leuprolide acetate (Viadur®, Lupron®,Leuprogel®, Eligard®), Mitoxantrone (Novantrone®, DHAD), Nilutamide(Nilandron®), Paclitaxel (Paxene®, Taxol®), Paclitaxel-DHA(Taxoprexin®), PC SPES, Prednisone, Triptorelin pamoate (TrelstarDepot®, Decapeptyl®), and Vinblastine (Velban®, VLB).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofprostate cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment ofprostate cancer and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Abarelix®) (Abarelix-Depot-M®, PPI-149, R-3827); AbirateroneAcetate® (CB-7598, CB-7630), ABT-627 (ET-1 inhibitor), APC-8015(Provenge®, Dendritic cell therapy), Avorelin® (Meterelin®, MF-6001,EP-23904), CEP-701 (KT-5555), CN-706, CT-2584 (Apra®, CT-2583, CT-2586,CT-3536), GBC-590, Globo H hexasaccharide (Globo H-KLH®), Interferonalpha 2a (Intron A®), Liarozole (Liazal, Liazol, R-75251, R-85246,Ro-85264), MDX-447 (MDX-220, BAB-447, EMD-82633, H-447,anti-EGFr/FcGammaR1r), NPI-0052 (proteasome inhibitor), OncoVAX-P(OncoVAX-PrPSA), PROSTVAC, PS-341 (LDP-341, 26S proteasome inhibitor),PSMA MAb (Prostate Specific Membrane Antigen monoclonal antibody), andR-flurbiprofen (Flurizan®, E-7869, MPC-7869).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofprostate cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofprostate cancer and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Docetaxel+Estramustine, Mitoxantrone+Hydrocortisone,Mitoxantrone+Prednisone, Navelbine+Estramustine,Paclitaxel+Estramustine, and Vinblastine+Estramustine.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of prostate cancers and premalignant conditions.

In further specific embodiments, polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent pancreatic cancer and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent pancreatic cancer andpremalignant conditions. Pancreatic cancers which may be treated usingpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to,adenocarcinoma, endocrine (islet cell) tumors, tumors confined to thepancreas, locally advanced pancreatic cancer and metastatic pancreaticcancer.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent pancreatic cancerand premalignant conditions. Agonists and/or antagonists of the presentinvention may be used in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent pancreatic cancer and premalignant conditions. Pancreaticcancers which may be treated using agonists and/or antagonists of thepresent invention include, but are not limited to, adenocarcinoma,endocrine (islet cell) tumors, tumors confined to the pancreas, locallyadvanced pancreatic cancer and metastatic pancreatic cancer.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat locally advanced pancreatic cancer. In other preferredembodiments, agonists and/or antagonists of the invention are used totreat metastatic pancreatic cancer and premalignant conditions.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered: in combination with oneor more surgical and/or radiological procedures useful in the treatmentof pancreatic cancer and premalignant conditions including, but notlimited to, pancreaticoduodenumectomy (Whipple resection).

In preferred embodiments, agonists and/or antagonists of the presentinvention may be administered in combination with one or more surgicaland/or radiological procedures useful in the treatment of pancreaticcancer and premalignant conditions including, but not limited to,pancreaticoduodenumectomy (Whipple resection).

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of pancreatic cancerand premalignant conditions including, but not limited to, Capecitabine(Xeloda®, Doxifluridine®, oral 5-FU), Cisplatin (Platinol®, CDDP),Fluorouracil (5FU, Adrucil®, Fluoroplex®, Efudex®), Gemcitabine (Gemto®,Gemzar®), and Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofpancreatic cancers.

Preferred combinations of therapeutic agents useful in the treatment ofpancreatic cancer and premalignant conditions which may be administeredin combination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Cisplatin+Gemcitabine, CP-358774+Gemcitabine, Docetaxel+Gemcitabine,Irinotecan+Fluorouracil, Irinotecan+Gemcitabine, andPaclitaxel+Gemcitabine.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of pancreatic cancers and premalignant conditions.

Further examples of therapeutic agents useful in the treatment ofpancreatic cancer and premalignant conditions which may be administeredin combination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, ABX-EGF (anti-EGFr MAb), Acetyldinaline (CI-994, GOE-5549, GOR-5549,PD-130636), BMS-214662 (BMS-192331, BMS-193269, BMS-206635), BNP-1350(BNPI-1100, Karenitecins), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb,Cetuximab®), C242-DM1 (huC242-DM1, SB-408075), Carbendazin®(FB-642),Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), CMT-3 (COL-3,Metastat®), CP-358774 (Tarceva®, OSI-774, EGFR inhibitor), Docetaxel(Taxotere®, Taxane®), Exetecan mesylate (DX-8951, DX-8951f),Flavopiridol (HMR-1275), Gastrimmune® (Anti-gastrin-17 immunogen,anti-g17), GBC-590, Herceptin® (Trastuzumab®, Anti-HER-2 monoclonalantibody, Anti-EGFR-2 MAb), HSPPC-96 (HSP cancer vaccine, gp96 heatshock protein-peptide complex), Irofulven (MGI-114), ISIS-2503 (Rasantisense), Onyx-015 (p53 gene therapy), Paclitaxel (Paxene®), Taxol®),Pemetrexed disodium (Alimta®, MTA, multitargeted antifolate, LY 231514),Perillyl alcohol (perilla alcohol, perillic alcohol, perillol,NSC-641066), RFS-2000 (9-nitrocamptothecan, 9-NC, Rubitecan®), andRituximab® (Rituxan®, anti-CD20 MAb).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofpancreatic cancers and premalignant conditions.

In further particular embodiments, polynucleotides and/or polypeptidesof the invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent hepatic cancer and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent hepatic cancer andpremalignant conditions. Hepatic cancers which may be treated usingpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to,hepatocellular carcinoma, malignant hepatoma, cholangiocarcinoma, mixedhepatocellular cholangiocarcinoma or hepatoblastoma.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent hepatic cancerand premalignant conditions. Agonists and/or antagonists of the presentinvention may be used in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent hepatic cancer and premalignant conditions. Hepaticcancers which may be treated using agonists and/or antagonists of thepresent invention include, but are not limited to, hepatocellularcarcinoma, malignant hepatoma, cholangiocarcinoma, mixed hepatocellularcholangiocarcinoma or hepatoblastoma.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat hepatoblastoma. In other preferred embodiments,agonists and/or antagonists of the invention are used to treathepatocellular carcinoma.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more surgical and/or radiological procedures useful in the treatmentof hepatic cancers and premalignant conditions including, but notlimited to, partial hepatectomy, liver transplant, radiofrequencyablation, laser therapy, microwave therapy, cryosurgery, percutaneousethanol injection, hepatic arterial infusion, hepatic artery ligation,chemoembolization and external beam radiation therapy.

In preferred embodiments, agonists and/or antagonists of the presentinvention may be administered in combination with one or more surgicaland/or radiological procedures useful in the treatment of hepaticcancers and premalignant conditions including, but not limited to,partial hepatectomy, liver transplant, radiofrequency ablation, lasertherapy, microwave therapy, cryosurgery, percutaneous ethanol injection,hepatic arterial infusion, hepatic artery ligation, chemoembolizationand external beam radiation therapy.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of hepatic cancer andpremalignant conditions including, but not limited to, Aldesleukin(IL-2, Proleukin®), Cisplatin (Platinol®, CDDP), Doxorubicin(Adriamycin®, Doxil®, Rubex®), Etoposide phosphate (Etopophos®),Etoposide (VP-16, Vepesid®), Fluorouracil (5-FU, Adrucil®, Fluoroplex®,Efudex®), I-131 Lipidiol®, Ifosfamide (IFEX®), Megestrol acetate(Megace®, Pallace®), Pravastatin sodium (Pravachol®), and Vincristine(Oncovin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofhepatic cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofhepatic cancer and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Cisplatin+Doxorubicin, Cisplatin+Etoposide,Cisplatin+Vincristine+Fluorouracil, andIfosfamide+Cisplatin+Doxorubicin.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of hepatic cancers and premalignant conditions.

In further particular embodiments, polynucleotides and/or polypeptidesof the invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent ovarian cancer and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent ovarian cancer andpremalignant conditions. Ovarian cancers which may be treated usingpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to, epithelialcarcinoma, germ cell tumors and stromal tumors.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent ovarian cancerand premalignant conditions. Agonists and/or antagonists of the presentinvention may be used in combination with one or more surgical and/orradiological procedures and/or therapeutic agents to treat, ameliorateand/or prevent ovarian cancer and premalignant conditions. Ovariancancers which may be treated using agonists and/or antagonists of thepresent invention include, but are not limited to, epithelial carcinoma,germ cell tumors and stromal tumors.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat germ cell tumors and premalignant conditions. In otherpreferred embodiments, agonists and/or antagonists of the invention areused to treat epithelial carcinoma and premalignant conditions.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more surgical and/or radiological procedures useful in the treatmentof ovarian cancer and premalignant conditions including, but not limitedto, hysterectomy, oophorectomy, hysterectomy with bilateralsalpingo-oophorectomy, omentectomy, tumor debulking, external beamradiation therapy and intraperitoneal radiation therapy.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedsurgical and/or radiological procedures in the treatment, ameliorationand/or prevention of ovarian cancers and premalignant conditions.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of ovarian cancer andpremalignant conditions including, but not limited to, Altretamine(Hexylen®, hexamethylmelamine, Hexastat®), Bleomycin (Blenoxane®),Carboplatin (Paraplatin®, CBDCA), Cisplatin (Platinol®, CDDP),Cyclophosphamide (Cytoxan®, Neosar®, CTX), Dactinomycin (Cosmegen®),Doxorubicin (Adriamycin®, Doxil®, Rubex®), Etoposide phosphate(Etopophos®), Etoposide (VP-16, Vepesid®), Fluorouracil (5-FU, Adrucil®,Fluoroplex®, Efudex®), Gemcitabine (Gemto®, Gemzar®), Ifosfamide(IFEX®), Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1),Leucovorin (Leucovorin®, Wellcovorin®), Melphalan (L-PAM, Alkeran®,Phenylalanine mustard), Paclitaxel (Paxene®, Taxol®), Tamoxifen(Nolvadex®, Vinblastine (Velban®, VLB) and Vincristine (Oncovin®, OncoTCS®, VCR, Leurocristine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofovarian cancers and premalignant conditions.

Preferred combinations of therapeutic agents useful in the treatment ofovarian cancer and premalignant conditions which may be administered incombination with polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof include, but are not limitedto, Bleomycin+Etoposide+Platinol® (Cisplatin) (BEP),Carboplatin+Cyclophosphamide, Carboplatin+Paclitaxel,Carboplatin+Etoposide+Bleomycin (CEB), Cisplatin+Cyclophosphamide,Cisplatin+Etoposide, Cisplatin+Paclitaxel,Cisplatin+Ifosfamide+Vinblastine, Fluorouracil+Leucovorin, Platinol®(Cisplatin)+Vinblastine+Bleomycin (PVB), andVincristine+Dactinomycin+Cyclophosphamide.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of ovarian cancers and premalignant conditions.

In further particular embodiments, polynucleotides and/or polypeptidesof the invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent Ewing's sarcoma. Polynucleotides and/orpolypeptides of the invention and/or agonists and/or antagonists thereofmay be used in combination with one or more surgical and/or radiologicalprocedures and/or therapeutic agents to treat, ameliorate and/or preventEwing's sarcoma. Ewing's sarcoma family tumors which may be treatedusing polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof include, but are not limited to,Ewing's tumor of bone (ETB), extraosseus Ewing's (EOE), primitiveneuroectodermal tumors (PNET or peripheral neuroepithelioma) and Askin'stumor.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent Ewing's sarcoma.Agonists and/or antagonists of the present invention may be used incombination with one or more surgical and/or radiological proceduresand/or therapeutic agents to treat, ameliorate and/or prevent Ewing'ssarcoma. Ewing's sarcoma family tumors which may be treated usingagonists and/or antagonists of the present invention include, but arenot limited to, Ewing's tumor of bone (ETB), extraosseus Ewing's (EOE),primitive neuroectodermal tumors (PNET or peripheral neuroepithelioma)and Askin's tumor.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat Ewing's tumor of bone. In other preferred embodiments,agonists and/or antagonists of the invention are used to treatperipheral neuroepithelioma.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more surgical and/or radiological procedures useful in the treatmentof Ewing's sarcoma family tumors.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more surgical and/orradiological procedures useful in the treatment of Ewing's sarcomafamily tumors.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of Ewing's sarcomafamily tumors including, but not limited to, Cyclophosphamide (Cytoxan®,Neosar®, CTX), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Etoposidephosphate (Etopophos®), Etoposide (VP-16, Vepesid®), Filgrastim(Neupogen®, G-CSF), Ifosfamide (IFEX®), Topotecan (Hycamtin®,SK&F-104864, NSC-609699, Evotopin®), and Vincristine (Oncovin®, OncoTCS®, VCR, Leurocristine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofEwing's sarcoma family tumors.

Preferred combinations of therapeutic agents useful in the treatment ofEwing's sarcoma family tumors which may be administered in combinationwith polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof include, but are not limited to,Cyclophosphamide+Topotecan, Cyclophosphamide+Doxorubicin+Vincristine,Cyclophosphamide+Doxorubicin+Vincristine, alternating withIfosfamide+Etoposide andcyclophosphamide+Doxorubicin+Vincristine,alternating with Filgrastim+Ifosfamide+Etoposide.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of Ewing's sarcoma family tumors.

In further specific embodiments, polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof are used totreat, ameliorate and/or prevent hematological cancers and premalignantconditions. Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof may be used in combination with oneor more surgical and/or radiological procedures and/or therapeuticagents to treat, ameliorate and/or prevent hematological cancers andpremalignant conditions. Hematological cancers which may be treatedusing polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof include, but are not limited to,non-Hodgkin's lymphoma (e.g., small lymphocytic lymphoma, follicularcenter cell lymphoma, lymphoplasmacytoid lymphoma, marginal zonelymphoma, mantle cell lymphoma, immunoblastic lymphoma, burkitt'slymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, anaplasticlarge cell lymphoma and intestinal T-cell lymphoma), leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia and plasma cellneoplasms including multiple myeloma.

In preferred embodiments, agonists and/or antagonists of the presentinvention are used to treat, ameliorate and/or prevent hematologicalcancers and premalignant conditions. Agonists and/or antagonists of thepresent invention may be used in combination with one or more surgicaland/or radiological procedures and/or therapeutic agents to treat,ameliorate and/or prevent hematological cancers and premalignantconditions. Hematological cancers which may be treated using agonistsand/or antagonists of the present invention include, but are not limitedto, non-Hodgkin's lymphoma (e.g., small lymphocytic lymphoma, follicularcenter cell lymphoma, lymphoplasmacytoid lymphoma, marginal zonelymphoma, mantle cell lymphoma, immunoblastic lymphoma, burkitt'slymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, anaplasticlarge cell lymphoma and intestinal T-cell lymphoma), leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia and plasma cellneoplasms including multiple myeloma.

In preferred embodiments, agonists and/or antagonists of the inventionare used to treat plasma cell neoplasms. In certain preferredembodiments, that plasma cell neoplasm is multiple myeloma.

In other preferred embodiment, agonists and/or antagonists of theinvention are used to treat non-Hodgkin's lymphoma.

In other preferred embodiments, agonists and/or antagonists of theinvention are used to treat leukemia. In certain preferred embodiments,that leukemia is acute lymphocytic leukemia. In certain preferredembodiments, that leukemia is chronic lymphocytic leukemia.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more surgical and/or radiological procedures useful in the treatmentof hematological cancer and premalignant conditions including, but notlimited to, bone marrow transplantation, external beam radiation andtotal body irradiation.

In specific embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more surgical and/orradiological procedures useful in the treatment of hematological cancerand premalignant conditions including, but not limited to, bone marrowtransplantation, external beam radiation and total body irradiation.

In preferred embodiments, agonists and/or antagonists of the presentinvention may be administered in combination with one or more surgicaland/or radiological procedures useful in the treatment of multiplemyeloma including, but not limited to, allogeneic bone marrowtransplantation and peripheral stem cell support.

In other preferred embodiments, agonists and/or antagonists of thepresent invention may be administered in combination with one or moresurgical and/or radiological procedures useful in the treatment ofnon-Hodgkin's lymphoma including, but not limited to, allogeneic bonemarrow transplantation and peripheral stem cell support.

In other preferred embodiments, agonists and/or antagonists of thepresent invention may be administered in combination with one or moresurgical and/or radiological procedures useful in the treatment ofleukemia including, but not limited to, allogeneic bone marrowtransplantation and peripheral stem cell support. In specificembodiments, agonists and/or antagonists of the invention are used totreat acute lymphocytic leukemia (ALL). In other specific embodiments,agonists and/or antagonists of the invention are used to treat chroniclymphocytic leukemia (CLL).

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of multiple myelomaincluding, but not limited to, Alkylating agents, Anthracyclines,Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Cyclophosphamide(Cytoxan®, Neosar®, CTX), Dexamethasone (Decadron®), Doxorubicin(Adriamycin®, Doxil®, Rubex®), Melphalan (L-PAM, Alkeran®, Phenylalaninemustard), Prednisone, Thalidomide and thalidomide analogs, including butnot limited to, lenalidomide (CC-5013, REVLIMID®) and CC-4047(ACTIMID™), and Vincristine (Oncovorin®, Onco TCS®, VCR,Leurocristine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofmultiple myeloma.

Preferred combinations of therapeutic agents useful in the treatment ofmultiple myeloma which may be administered in combination withpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to,Cyclophosphamide+Prednisone, Melphalan+Prednisone (MP),Vincristine+Adriamycin®+Dexamethasone (VAD),Vincristine+Carmustine+Melphalan+Cyclophosphamide+Prednisone (VBMCP; theM2 protocol), and Vincristine+Melphalan+Cyclophosphamide+Prednisonealternating with Vincristine+Carmustine+Doxorubicin+Prednisone(VMCP/VBAP).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of multiple myeloma.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of non-Hodgkin'slymphoma including, but not limited to, 2-chlorodeoxyadenosine,Amifostine (Ethyol®, Ethiofos®, WR-272), Bexarotene (Targretin®,Targretin Gel®, Targretin Oral®, LGD1069), Bleomycin (Blenoxane®),Busulfan (Busulfex®, Myleran®), Carboplatin (Paraplatin®, CBDCA),Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Chlorambucil(Leukeran®), Cisplatin (Platinol®, CDDP), Cladribine (2-CdA,Leustatin®), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine(Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®), Dacarbazine (DTIC),Daunorubicin (Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®),Denileukin diftitox (Ontak®), Dexamethasone (Decadron®), Dolasetronmesylate (Anzemet®), Doxorubicin (Adriamycin®, Doxil®, Rubex®),Erythropoietin (EPO®, Epogen®, Procrit®), Etoposide phosphate(Etopophos®), Etoposide (VP-16, Vepesid®), Fludarabine (Fludara®, FAMP),Granisetron (Kytril®), Hydrocortisone, Idarubicin (Idamycin®, DMDR,IDA), Ifosfamide (IFEX®), Interferon alpha (Alfaferone®, Alpha-IF®),Interferon alpha 2a (Intron A®), Mechlorethamine (Nitrogen Mustard, HN₂,Mustargen®), Melphalan (L-PAM, Alkeran®, Phenylalanine mustard),Methotrexate® (MTX, Mexate®, Folex®), Methylprednisolone (Solumedrol®),Mitoxantrone (Novantrone®, DHAD), Ondansetron (Zofran®), Pentostatin(Nipent®, 2-deoxycoformycin), Perfosfamide(4-hydroperoxycyclophosphamide, 4-HC), Prednisone, Procarbazine(Matulane®), Rituximab® (Rituxan®, anti-CD20 MAb), Thiotepa(triethylenethiophosphaoramide, Thioplex®), Topotecan (Hycamtin®,SK&F-104864, NSC-609699, Evotopin®), Vinblastine (Velban®, VLB),Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®) and Vindesine(Eldisine®, Fildesin®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofnon-Hodgkin's lymphoma.

Preferred combinations of therapeutic agents useful in the treatment ofnon-Hodgkin's lymphoma which may be administered in combination withpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to,Adriamycin®+Blenoxane+Vinblastine+Dacarbazine (ABVD), Anti-idiotypetherapy (BsAb)+Interferon alpha, Anti-idiotype therapy(BsAb)+Chlorambucil, Anti-idiotype therapy (BsAb)+Interleukin-2, BCNU(Carmustine)+Etoposide+Ara-C (Cytarabine)+Melphalen (BEAM),Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+Vincristine+Procarbazine+Prednisone(BEACOPP), Bryostatin+Vincristine, Cyclophosphamide+BCNU(Carmustine)+VP-16 (Etoposide) (CBV),Cyclophosphamide+Vincristine+Prednisone (CVP),Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine(Oncovorin)+Prednisone (CHOP), Cyclophosphamide+Novantrone®(Mitoxantrone)+Vincristine (Oncovorin)+Prednisone (CNOP),Cyclophosphamide+Doxorubicin+Teniposide+Prednisone,Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine(Oncovorin)+Prednisone+Rituximab (CHOP+Rituximab),Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon alpha,Cytarabine+Bleomycin+Vincristine+Methotrexate (CytaBOM),Dexamethasone+Cytarabine+Cisplatin (DHAP),Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE),Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+Prednisone(Stanford V), Etoposide+Vinblastine+Adriamycin (EVA),Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP),Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC), Fludarabine,Mitoxantrone+Dexamethasone (FMD), Fludarabine, Dexamethasone, Cytarabine(ara-C), +Cisplatin (Platinol®) (FluDAP), Ifosfamide+Cisplatin+Etoposide(ICE), Mechlorethamine+Oncovin® (Vincristine)+Procarbazine+Prednisone(MOPP), Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate withleucovorinrescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone(m-BACOD), Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide(ProMACE), Thiotepa+Busulfan+Cyclophosphamide,Thiotepa+Busulfan+Melphalan, Topotecan+Paclitaxel, and Vincristine(Oncovin®)+Adriamycin®+Dexamethasone (VAD).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of non-Hodgkin's lymphoma.

Further examples of therapeutic agents useful in the treatment ofnon-Hodgkin's lymphoma which may be administered in combination withpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to, A007(4-4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone), AG-2034(AG-2024, AG-2032, GARFT [glycinamide ribonucleoside transformylase]inhibitor), Aldesleukin (IL-2, Proleukin®), Alemtuzumab (Campath®),Alitretinoin (Panretin®, LGN-1057), Altretamine (Hexylen®,hexamethylmelamine, Hexastat®), Aminocamptothecin (9-AC,9-Aminocamptothecin, NSC 603071), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv,anti-NHL MAb), Anti-idiotype therapy (BsAb), Arabinosylguanine (Ara-G,GW506U78), Arsenic trioxide (Trisenox®, ATO), B43-Genistein (anti-CD19Ab/genistein conjugate), B7 antibody conjugates, Betathine (Beta-LT),BLyS antagonists, Bryostatin-1 (Bryostatin®, BMY-45618, NSC-339555),CHML (Cytotropic Heterogeneous Molecular Lipids), Clofarabine(chloro-fluoro-araA), Daclizumab (Zenapax®), Depsipeptide (FR901228,FK228), Dolastatin-10 (DOLA-10, NSC-376128), Epirubicin (Ellence®, EPI,4′ epi-doxorubicin), Epratuzumab (Lymphocide®, humanized anti-CD22,HAT), Fly3/flk2 ligand (Mobista®), G3139 (Genasense®, GentaAnticode®,Bcl-2 antisense), Hu1D10 (anti-HLA-DR MAb, SMART ID 10), HumaLYM(anti-CD20 MAb), Ibritumomab tiuxetan (Zevalin®), Interferon gamma(Gamma-interferon, Gamma 100®, Gamma-IF), Irinotecan (Camptosar®,CPT-11, Topotecin®, CaptoCPT-1), ISIS-2053, ISIS-3521 (PKC-alphaantisense), Lmb-2 immunotoxin (anti-CD25 recombinant immuno toxin,anti-Tac(Fv)-PE38), Leuvectin®(cytofectin+IL-2 gene, IL-2 gene therapy),Lym-1(131-I LYM-1), Lymphoma vaccine (Genitope), Nelarabine (Compound506, U78), Neugene compounds (Oncomyc-NG®, Resten-NG®, myc antisense),NovoMAb-G2 scFv (NovoMAb-G2 IgM), 06-benzylguanine (BG, Procept®),Oxaliplatin (Eloxatine®, Eloxatin®), Paclitaxel (Paxene®, Taxol®),Paclitaxel-DHA (Taxoprexin®), Peldesine (BCX-34, PNP inhibitor),Rebeccamycin and Rebeccamycin analogues, SCH-66336, Sobuzoxane (MST-16,Perazolin®), SU5416 (Semaxanib®, VEGF inhibitor), TER-286, Thalidomideand thalidomide analogs, including but not limited to, lenalidomide(CC-5013, REVLIMID®) and CC4047 (ACTIMID™), TNP-470 (AGM-1470),Tositumomab (Bexxar®), Valspodar (PSC 833), Vaxid (B-cell lymphoma DNAvaccine), Vinorelbine (Navelbine®), WF10 (macrophage regulator) andXR-9576 (XR-9351, P-glycoprotein/MDR inhibitor).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofnon-Hodgkin's lymphoma.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of acute lymphocyticleukemia including, but not limited to, Amsacrine, Carboplatin(Paraplatin®, CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®),Cholecaliferol, Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine(Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®), Daunorubicin(Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®), Dexamethasone(Decadron®), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Etoposide(VP-16, Vepesid®), Filgrastam® (Neupogen®, G-CSF, Leukine®), Fludarabine(Fludara®, FAMP), Idarubicin (Idamycin®, DMDR, IDA), Ifosfamide (IFEX®),Imatinib mesylate (STI-571, Imatinib®), Glivec®, Gleevec®, Ab1 tyrosinekinase inhibitor), Interferon gamma (Gamma-interferon, Gamma 100®,Gamma-IF), L-asparaginase (Elspar®, Crastinin®, Asparaginase Medac®,Kidrolase®), Mercaptopurine (6-mercaptopurine, 6-MP), Methotrexate®(MTX, Mexate®, Folex®), Mitoxantrone (Novantrone®, DHAD), Pegaspargase®(Oncospar®), Prednisone, Retinoic acid, Teniposide (VM-26, Vumon®),Thioguanine (6-thioguanine, 6-TG), Topotecan (Hycamtin®, SK&F-104864,NSC-609699, Evotopin®), Tretinoin (Retin-A®, Atragen®), ATRA, Vesanoid®)and Vincristine (Oncovorin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofacute lymphocytic leukemia.

Further examples of therapeutic agents useful in the treatment of acutelymphocytic leukemia which may be administered in combination withpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to,Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminopterin,Annamycin (AR-522, annamycin LF, Aronex®), Arabinosylguanine (Ara-G,GW506U78, Nelzarabine®), Arsenic trioxide (Trisenox®, ATO, Atrivex®),B43-Genistein (anti-CD19 Ab/genistein conjugate), B43-PAP (anti-CD19Ab/pokeweed antiviral protein conjugate), Cordycepin, CS-682, Decitabine(5-aza-2′-deoxyytidine), Dolastatin-10 (DOLA-10, NSC-376128), G3139(Genasense®, GentaAnticode®, Bcl-2 antisense), Irofulven (MGI-114,Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Quinine,TNP-470 (AGM-1470, Fumagillin), Trimetrexate (Neutrexin®), Troxacitabine(BCH-204, BCH-4556, Troxatyl®), UCN-01 (7-hydroxystaurosporine),WHI-P131 and WT1 Vaccine.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofacute lymphocytic leukemia.

Preferred combinations of therapeutic agents useful in the treatment ofacute lymphocytic leukemia which may be administered in combination withpolynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof include, but are not limited to,Carboplatin+Mitoxantrone, Carmustine+Cyclophosphamide+Etoposide,Cytarabine+Daunorubicin, Cytarabine+Doxorubicin, Cytarabine+Idarubicin,Cytarabine+Interferon gamma, Cytarabine+L-asparaginase,Cytarabine+Mitoxantrone, Cytarabine+Fludarabine and Mitoxantrone,Etoposide+Cytarabine, Etoposide+Ifosfamide, Etoposide+Mitoxantrone,Ifosfamide+Etoposide+Mitoxantrone, Ifosfamide+Teniposide,Methotrexate+Mercaptopurine,Methotrexate+Mercaptopurine+Vincristine+Prednisone,Phenylbutyrate+Cytarabine, Phenylbutyrate+Etoposide,Phenylbutyrate+Topotecan, Phenylbutyrate+Tretinoin, Quinine+Doxorubicin,Quinine+Mitoxantrone+Cytarabine, Thioguanine+Cytarabine+Amsacrine,Thioguanine+Etoposide+Idarubicin, Thioguanine+Retinoicacid+Cholecaliferol, Vincristine+Prednisone, Vincristine+Prednisone andL-asparaginase,Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin,Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Filgrastim,Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate,andVincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate+Filgrastim.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of acute lymphocytic leukemia.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof may be administered in combination with oneor more therapeutic agents useful in the treatment of chroniclymphocytic leukemia including, but not limited to, Chlorambucil(Leukeran®), Cladribine (2-CdA, Leustatin®), Cyclophosphamide (Cytoxan®,Neosar®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside,DepoCyt®, cytarabine ocfosfate, ara-CMP), Doxorubicin (Adriamycin®,Doxil®, Rubex®), Fludarabine (Fludara®, FAMP), Pentostatin (Nipent®,2-deoxycoformycin), Prednisone and Vincristine (Oncovorin®, Onco TCS®,VCR, Leurocristine®).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofchronic lymphocytic leukemia.

Further examples of therapeutic agents useful in the treatment ofchronic lymphocytic leukemia which may be administered in combinationwith polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof include, but are not limited to,Alemtuzumab (Campath®), Aminocamptothecin (9-AC, 9-Aminocamptothecin,NSC 603071), Aminopterin, Annamycin (AR-522, annamycin LF, Aronex®),Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®, Compound 506U78),Arsenic trioxide (Trisenox®, ATO, Atrivex®), Bryostatin-1 (Bryostatin®,BMY-45618, NSC-339555), CS-682, Dolastatin-10 (DOLA-10, NSC-376128),Filgrastim (Neupogen®, G-CSF, Leukine), Flavopiridol (NSC-649890,HMR-1275), G3139 (Genasense®, GentaAnticode®, Bcl-2 antisense),Irofulven (MGI-114, Ivofulvan, Acylfulvene analogue), MS-209,Phenylbutyrate, Rituximab®(Rituxan®, anti-CD20 MAb), Thalidomide andthalidomide analogs, including but not limited to, lenalidomide(CC-5013, REVLIMID®) and CC-4047 (ACTIMID™), Theophylline, TNP-470(AGM-1470, Fumagillin), UCN-01 (7-hydroxystaurosporine) and WHI-P131.

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agents in the treatment, amelioration and/or prevention ofchronic lymphocytic leukemia.

Preferred combinations of therapeutic agents useful in the treatment ofchronic lymphocytic leukemia which may be administered in combinationwith polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof include, but are not limited to,Fludarabine+Prednisone, andCyclophosphamide+Doxorubicin+Vincristine+Prednisone (CHOP).

In preferred embodiments, agonists and/or antagonists of the inventionare administered in combination with one or more of the above-describedtherapeutic agent combinations in the treatment, amelioration and/orprevention of chronic lymphocytic leukemia.

Diseases associated with increased apoptosis include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration); and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia. In preferred embodiments, DR4 polynucleotides,polypeptides and/or agonists are used to treat the diseases anddisorders listed above.

Many of the pathologies associated with HIV are mediated by apoptosis,including HIV-induced nephropathy and HIV encephalitis. Thus, inadditional preferred embodiments, DR4 polynucleotides, polypeptides,and/or DR4 agonists of the invention are used to treat and/or preventAIDS and pathologies associated with AIDS. Another embodiment of thepresent invention is directed to the use of DR4 to reduce TRAIL-mediateddeath of T-cells in HIV-infected patients.

The state of immunodeficiency that defines AIDS is secondary to adecrease in the number and function of CD4⁺ T-lymphocytes. Recentreports estimate the daily loss of CD4⁺ T-cells to be between 3.5×10⁷and 2×10⁹ cells (Wei X., et al., Nature 373:117-122 (1995)). One causeof CD4⁺ T-cell depletion in the setting of HIV infection is believed tobe HIV-induced apoptosis (see, for example, Meyaard et al., Science257:217-219, (1992); Groux et al., J Exp. Med., 175:331, (1992); andOyaizu et al., in Cell Activation and Apoptosis in HIV Infection,Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-114).Indeed, HIV-induced apoptotic cell death has been demonstrated not onlyin vitro but also, more importantly, in infected individuals (Ameisen,J. C., AIDS 8:1197-1213 (1994); Finkel, T. H., and Banda, N. K., Curr.Opin. Immunol. 6:605-615 (1995); Muro-Cacho, C. A. et al., J. Immunol.154:5555-5566 (1995)). Furthermore, apoptosis and CD4⁺ T-lymphocytedepletion is tightly correlated in different animal models of AIDS(Brunner, T., et al., Nature 373:441-444 (1995); Gougeon, M. L., et al.,AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis is notobserved in those animal models in which viral replication does notresult in AIDS (Gougeon, M. L. et al., AIDS Res. Hum. Retroviruses9:553-563 (1993)). Further data indicates that uninfected but primed oractivated T lymphocytes from HIV-infected individuals undergo apoptosisafter encountering the TNF-family ligand FasL. Using monocytic celllines that result in death following HIV infection, it has beendemonstrated that infection of U937 cells with HIV results in the denovo expression of FasL and that FasL mediates HIV-induced apoptosis(Badley, A. D. et al., J. Virol. 70:199-206 (1996)). Further theTNF-family ligand was detectable in uninfected macrophages and itsexpression was upregulated following HIV infection resulting inselective killing of uninfected CD4 T-lymphocytes (Badley, A. D et al.,J. Virol. 70:199-206 (1996)). Further, additional studies haveimplicated Fas-mediated apoptosis in the loss of T-cells in HIVindividuals (Katsikis et al., J. Exp. Med. 181:2029-2036 (1995)). It isalso possible that T-cell apoptosis occurs through multiple mechanisms.Further, at least some of the T-cell death seen in HIV patients may bemediated by TRAIL.

Thus, by the invention, a method for treating and/or preventing HIV⁺individuals is provided which involves administering DR4, DR4polypeptides, polynucleotides, antagonists, and/or agonists of thepresent invention to reduce selective killing of CD4⁺ T-lymphocytes.While not wanting to be bound by theory, activated human T-cells arebelieved to be induced to undergo programmed cell death (apoptosis) upontriggering through the CD3/T-cell receptor complex, a process termedactivated-induced cell death (AICD). AICD of CD4⁺ T-cells isolated fromHIV-Infected asymptomatic individuals has been reported (Groux et al.,supra). Thus, AICD may play a role in the depletion of CD4⁺ T-cells andthe progression to AIDS in HIV-infected individuals. Thus, the presentinvention provides a method of inhibiting TRAIL-mediated T-cell death inHIV patients, comprising administering a DR4 polypeptide of theinvention (preferably, a soluble DR4 polypeptide) and/or DR4 antagonistof the invention to the patients. Modes of administration and dosagesare discussed in detail below. In one embodiment, the patient isasymptomatic when treatment with DR4 commences. If desired, prior totreatment, peripheral blood T-cells may be extracted from an HIVpatient, and tested for susceptibility to TRAIL-mediated cell death byprocedures known in the art. In one embodiment, a patient's blood orplasma is contacted with DR4 polypeptides of the invention ex vivo. TheDR4 polypeptides of the invention may be bound to a suitablechromatography matrix by procedures known in the art. The patient'sblood or plasma flows through a chromatography column containing DR4bound to the matrix, before being returned to the patient. Theimmobilized DR4 polypeptide binds TRAIL, thus removing TRAIL proteinfrom the patient's blood.

In additional embodiments a DR4 polypeptide and/or antagonist of theinvention is administered in combination with other inhibitors of T-cellapoptosis. For example, as discussed above, Fas-mediated apoptosis alsohas been implicated in loss of T-cells in HIV individuals (Katsikis etal., J. Exp. Med. 181:2029-2036, 1995). Thus, a patient susceptible toboth Fas ligand mediated and TRAIL mediated T-cell death may be treatedwith both an agent that blocks TRAIL/TRAIL receptor interactions and anagent that blocks Fas-ligand/Fas interactions. Suitable agents forblocking binding of Fas-ligand to Fas include, but are not limited to,soluble Fas polypeptides; multimeric forms of soluble Fas polypeptides(e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas withouttransducing the biological signal that results in apoptosis;anti-Fas-ligand antibodies that block binding of Fas-ligand to Fas; andmuteins of Fas-ligand that bind Fas but do not transduce the biologicalsignal that results in apoptosis. Preferably, the antibodies employedaccording to this method are monoclonal antibodies. Examples of suitableagents for blocking Fas-ligand/Fas interactions, including blockinganti-Fas monoclonal antibodies, are described in Internationalapplication publication number WO 95/10540, hereby incorporated byreference.

Suitable agents, which also block binding of TRAIL to a TRAIL receptorthat may be administered with the polynucleotides and/or polypeptides ofthe present invention include, but are not limited to, soluble TRAILreceptor polypeptides (e.g., a soluble form of OPG, TR5 (Internationalapplication publication number WO 98/30693); DR5 (Internationalapplication publication number WO 98/41629); and TR10 (Internationalapplication publication number WO 98/54202)); multimeric forms ofsoluble TRAIL receptor polypeptides; and TRAIL receptor antibodies thatbind the TRAIL receptor without transducing the biological signal thatresults in apoptosis, anti-TRAIL antibodies that block binding of TRAILto one or more TRAIL receptors, and muteins of TRAIL that bind TRAILreceptors but do not transduce the biological signal that results inapoptosis. Preferably, the antibodies employed according to this methodare monoclonal antibodies.

In rejection of an allograft, the immune system of the recipient animalhas not previously been primed to respond because the immune system forthe most part is only primed by environmental antigens. Tissues fromother members of the same species have not been presented in the sameway that, for example, viruses and bacteria have been presented. In thecase of allograft rejection, immunosuppressive regimens are designed toprevent the immune system from reaching the effector stage. However, theimmune profile of xenograft rejection may resemble disease recurrencemore that allograft rejection. In the case of disease recurrence, theimmune system has already been activated, as evidenced by destruction ofthe native islet cells. Therefore, in disease recurrence the immunesystem is already at the effector stage. DR4 polynucleotides,polypeptides and/or agonists of the present invention are able tosuppress the immune response to both allografts and xenografts becauselymphocytes activated and differentiated into effector cells willexpress the DR4 polypeptide, and thereby are susceptible to compoundswhich enhance apoptosis. Thus, the present invention further provides amethod for creating immune privileged tissues. Antagonist of theinvention can further be used in the treatment and/or prevention ofInflammatory Bowel-Disease.

DR4 antagonists or agonists of the invention may be useful for treatingand/or preventing inflammatory diseases, such as rheumatoid arthritis,osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.

In addition, due to lymphoblast expression of DR4, soluble DR4, agonistor antagonist monoclonal antibodies may be used to treat and/or preventthis form of cancer. Further, soluble DR4 or neutralizing monoclonalantibodies may be used to treat and/or prevent various chronic and acuteforms of inflammation such as rheumatoid arthritis, osteoarthritis,psoriasis, septicemia, and inflammatory bowel disease.

In one embodiment, DR4 polypeptides, polynucleotides, and/or antagonistsof the invention may be used to treat and/or prevent cardiovasculardisorders, including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

In one embodiment, DR4 polynucleotides, polypeptides and/or antagonistsof the invention is used to treat and/or prevent thromboticmicroangiopathies. One such disorder is thrombotic thrombocytopenicpurpura (TTP) (Kwaan, H. C., Semin. Hematol. 24:71 (1987); Thompson etal., Blood 80:1890 (1992)). Increasing TTP-associated mortality rateshave been reported by the U.S. Centers for Disease Control (Torok etal., Am. J. Hematol. 50:84 (1995)). Plasma from patients afflicted withTTP (including HIV+ and HIV− patients) induces apoptosis of humanendothelial cells of dermal microvascular origin, but not large vesselorigin (Laurence et al., Blood 87:3245 (1996)). Plasma of TTP patientsthus is thought to contain one or more factors that directly orindirectly induce apoptosis. As described in International patentapplication number WO 97/01633 (hereby incorporated by reference), TRAILis present in the serum of TTP patients, and is likely to play a role ininducing apoptosis of microvascular endothelial cells. Anotherthrombotic microangiopathy is hemolytic-uremic syndrome (HUS) (Moake, J.L., Lancet, 343:393 (1994); Melnyk et al., (Arch. Intern. Med., 155:2077(1995); Thompson et al., supra). Thus, in one embodiment, the inventionis directed to use of DR4 to treat and/or prevent the condition that isoften referred to as “adult HUS” (even though it can strike children aswell). A disorder known as childhood/diarrhea-associated HUS differs inetiology from adult HUS. In another embodiment, conditions characterizedby clotting of small blood vessels may be treated and/or prevented usingDR4. Such conditions include, but are not limited to, those describedherein. For example, cardiac problems seen in about 5-10% of pediatricAIDS patients are believed to involve clotting of small blood vessels.Breakdown of the microvasculature in the heart has been reported inmultiple sclerosis patients. As a further example, treatment and/orprevention of systemic lupus erythematosus (SLE) is contemplated. In oneembodiment, a patient's blood or plasma is contacted with DR4polynucleotides and/or polypeptides of the invention ex vivo. The DR4polynucleotides and/or polypeptides of the invention may be bound to asuitable chromatography matrix by procedures known in the art. Accordingto this embodiment, the patient's blood or plasma flows through achromatography column containing DR4 polynucleotides and/or polypeptidesof the invention bound to the matrix, before being returned to thepatient. The immobilized DR4 binds TRAIL, thus removing TRAIL proteinfrom the patient's blood. Alternatively, DR4 polynucleotides and/orpolypeptides of the invention may be administered in vivo to a patientafflicted with a thrombotic microangiopathy. In one embodiment, asoluble form of DR4 polypeptide of the invention is administered to thepatient. Thus, the present invention provides a method for treatingand/or preventing a thrombotic microangiopathy, involving use of aneffective amount of DR4. A DR4 polypeptide may be employed in in vivo orex vivo procedures, to inhibit TRAIL-mediated damage to (e.g., apoptosisof) microvascular endothelial cells.

DR4 polynucleotides and/or polypeptides of the invention may be employedin combination with other agents useful in treating and/or preventing aparticular disorder. For example, in an in vitro study reported byLaurence et al. (Blood 87:3245 (1996)), some reduction of TTPplasma-mediated apoptosis of microvascular endothelial cells wasachieved by using an anti-Fas blocking antibody, aurintricarboxylicacid, or normal plasma depleted of cryoprecipitate. Thus, a patient maybe treated with a polynucleotide and/or polypeptide of the invention incombination with an agent that inhibits Fas-ligand-mediated apoptosis ofendothelial cells, such as, for example, an agent described above. Inone embodiment, DR4 polynucleotides and/or polypeptides of the inventionand an anti-FAS blocking antibody are both administered to a patientafflicted with a disorder characterized by thrombotic microangiopathy,such as TTP or HUS. Examples of blocking monoclonal antibodies directedagainst Fas antigen (CD95) are described in International patentapplication publication number WO 95/10540, hereby incorporated byreference.

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate (Rastinejad et al., Cell 56:345-355 (1989)). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., JMicrovasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment and/or prevention ofdiseases or disorders associated with neovascularization byadministration of the DR4 polynucleotides and/or polypeptides of theinvention (including DR4 agonists and/or antagonists). Malignant andmetastatic conditions which can be treated and/or prevented with thepolynucleotides and polypeptides of the invention include, but are notlimited to those malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)).

Additionally, ocular disorders associated with neovascularization whichcan be treated and/or prevented with the DR4 polynucleotides andpolypeptides of the present invention (including DR4 agonists and DR4antagonists) include, but are not limited to: neovascular glaucoma,diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

Additionally, disorders which can be treated and/or prevented with theDR4 polynucleotides and polypeptides of the present invention (includingDR4 agonists and DR4 antagonists) include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in the diagnosis, prognosis,treatment and/or prevention of a wide range of diseases and/orconditions. Such diseases and conditions include, but are not limitedto, cancer (e.g., immune cell related cancers, breast cancer, prostatecancer, ovarian cancer, follicular lymphoma, cancer associated withmutation or alteration of p53, brain tumor, bladder cancer,uterocervical cancer, colon cancer, colorectal cancer, non-small cellcarcinoma of the lung, small cell carcinoma of the lung, stomach cancer,etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial(e.g., viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2infection, herpesvirus infection (including, but not limited to, HSV-1,HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirusinfection, human papilloma virus infection, hepatitis infection (e.g.,HAV, HBV, HCV, etc.), Helicobacter pylori infection, invasiveStaphylococcia, etc.), parasitic infection, nephritis, bone disease(e.g., osteoporosis), atherosclerosis, pain, cardiovascular disorders(e.g., neovascularization, hypovascularization or reduced circulation(e.g., ischemic disease (e.g., myocardial infarction, stroke, etc.))),AIDS, allergy, inflammation, neurodegenerative disease (e.g.,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,pigmentary retinitis, cerebellar degeneration, etc.), graft rejection(acute and chronic), graft vs. host disease, diseases due toosteomyelodysplasia (e.g., aplastic anemia, etc.), joint tissuedestruction in rheumatism, liver disease (e.g., acute and chronichepatitis, liver injury, and cirrhosis), autoimmune disease (e.g.,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,immune complex glomerulonephritis, autoimmune diabetes, autoimmunethrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis,etc.), cardiomyopathy (e.g., dilated cardiomyopathy), diabetes, diabeticcomplications (e.g., diabetic nephropathy, diabetic neuropathy, diabeticretinopathy), influenza, asthma, psoriasis, glomerulonephritis, septicshock, and ulcerative colitis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis, woundhealing (e.g., wounds, burns, and bone fractures).

DR4 polynucleotides or polypeptides, or agonists of DR4, can be used inthe treatment and/or prevention of infectious agents. For example, byincreasing the immune response, particularly increasing theproliferation and differentiation of B-cells in response to aninfectious agent, infectious diseases may be treated and/or prevented.The immune response may be increased by either enhancing an existingimmune response, or by initiating a new immune response. Alternatively,DR4 polynucleotides or polypeptides, or agonists or antagonists of DR4,may also directly inhibit the infectious agent, without necessarilyeliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated and/or prevented by DR4 polynucleotides orpolypeptides, or agonists of DR4. Examples of viruses, include, but arenot limited to the following DNA and RNA viruses and viral families:Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae,Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue virus,HIV-1, HIV-2, Flaviviridae, Hepadnaviridae (e.g., hepatitis B virus),Herpesviridae (e.g., cytomegalovirus, herpes simplex viruses 1 and 2,varicella-zoster virus, Epstein-Barr virus (EBV), herpes B virus, andhuman herpes viruses 6, 7, and 8), Morbillivirus, Rhabdoviridae (e.g.,rabies virus), Orthomyxoviridae (e.g., influenza A virus, and influenzaB), Paramyxoviridae (e.g., parainfluenza virus), papilloma virus,Papovaviridae, Parvoviridae, Picornaviridae (e.g., EMCV and poliovirus),Poxyiridae (e.g., areola or vaccinia virus), Reoviridae (e.g.,rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae(e.g., Rubivirus). These viruses and virus families can cause a varietyof diseases or symptoms, including, but not limited to: arthritis,bronchiollitis, respiratory diseases, encephalitis, eye infections(e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis(A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,Chikungunya, Rift Valley fever, yellow fever, meningitis, smallpox,opportunistic infections (e.g., AIDS, Kaposi's sarcoma), pneumonia,Burkitt's lymphoma, chickenpox, zoster, hemorrhagic fever, measles,mumps, parainfluenza, rabies, the common cold, polio, leukemia, rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. DR4 polynucleotides or polypeptides, or agonists orantagonists of DR4, can be used to treat, prevent, and/or detect any ofthese symptoms or diseases.

In specific embodiments, DR4 polynucleotides, polypeptides, or agonistsand/or antagonists are used to treat and/or prevent: meningitis, Dengue,EBV, and/or hepatitis.

In an additional specific embodiment DR4 polynucleotides, polypeptides,or agonists and/or antagonists are used to treat patients non-responsiveto one or more other commercially available hepatitis vaccines.

In a further specific embodiment, DR4 polynucleotides, polypeptides, oragonists and/or antagonists are used to treat AIDS.

Similarly, bacteria and fungi that can cause disease or symptoms andthat can be treated and/or prevented by DR4 polynucleotides orpolypeptides, or agonists or antagonists of DR4, include, but are notlimited to the following organisms. Bacteria include, but are notlimited to Actinomyces, Bacillus (e.g., B. anthracis), Bacteroides,Bordetella, Bartonella, Borrelia (e.g., B. burgdorferi), Brucella,Campylobacter, Capnocytophaga, Chlamydia, Clostridium, Corynebacterium,Coxiella, Dermatophilus, Enterococcus, Ehrlichia, Escherichia (e.g.,Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Francisella,Fusobacterium, Haemobartonella, Haemophilus (e.g., H. influenzae typeb), Helicobacter, Klebsiella, L-form bacteria, Legionella, Leptospira,Listeria, Mycobacteria (e.g., M. leprae and M. tuberculosis),Mycoplasma, Neisseria (e.g., N. gonorrheae and N. meningitidis),Neorickettsia, Nocardia, Pasteurella, Peptococcus, Peptostreptococcus,Pneumococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea, Salmonella(e.g., S. typhimurium and S. typhi), Serratia, Shigella, Staphylococcus(e.g., S. aureus), Streptococcus (e.g., S. pyogenes, S. pneumoniae, andGroup B streptococcus), Streptomyces, Treponema, Vibrio (e.g., Vibriocholerae) and Yersinia (e.g., Y. pestis). Fungi include, but are notlimited to: Absidia, Acremonium, Alternaria, Aspergillus, Basidiobolus,Bipolaris, Blastomyces, Candida (e.g., C. albicans), Coccidioides,Conidiobolus, Cryptococcus (e.g., C. neoformans), Curvalaria,Erysipelothrix, Epidermophyton, Exophiala, Geotrichum, Histoplasma,Madurella, Malassezia, Microsporum, Moniliella, Mortierella, Mucor,Paecilomyces, Penicillium, Phialemonium, Phialophora, Prototheca,Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus,Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon,and Xylohypha. These and other bacteria or fungi can cause diseases orsymptoms including, but not limited to: bacteremia, endocarditis, eyeinfections (conjunctivitis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as whooping cough or emphysema, sepsis, Lyme Disease,cat-scratch disease, dysentery, paratyphoid fever, food poisoning,typhoid, pneumonia, gonorrhea, meningitis, chlamydia, syphilis,diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism,gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexuallytransmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses),toxemia, urinary tract infections, and wound infections. DR4polynucleotides or polypeptides, or agonists or antagonists of DR4, canbe used to treat, prevent and/or detect any of these symptoms ordiseases.

In specific embodiments, DR4 polynucleotides, polypeptides, or agonistsand/or antagonists thereof are used to treat and/or prevent: tetanus,diphtheria, botulism, and/or meningitis type B.

Moreover, parasites causing parasitic diseases or symptoms that can betreated and/or prevented by DR4 polynucleotides or polypeptides, oragonists of DR4, include, but are not limited to: protozoan parasitesincluding, but not limited to, Babesia, Balantidium, Besnoitia,Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia,Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora,Nosema, Pentatrichomonas, Plasmodium (e.g., Plasmodium virax, Plasmodiumfalciparium, Plasmodium malariae and Plasmodium ovale), Pneumocystis,Sarcocystis, Schistosoma, Theileria, Toxoplasma, and Trypanosoma; andhelminth parasites including, but not limited to, Acanthocheilonema,Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia,Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus,Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria,Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa,Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus,Oesophagostomum, Onchocerca, Opisthorchis, Ostertagia, Parafilaria,Paragonimus, Parascaris, Physaloptera, Protostrongylus, Setaria,Spirocerca, Spirometra, Stephanofilaria, Strongyloides, Strongylus,Thelazia, Toxascaris, Toxocara, Trichinella, Trichostrongylus,Trichuris, Uncinaria, and Wuchereria. These parasites can cause avariety of diseases or symptoms, including, but not limited to: scabies,trombiculiasis, eye infections (e.g., river blindness), elephantiasis,intestinal disease (e.g., dysentery, giardiasis), liver disease, lungdisease, opportunistic infections (e.g., AIDS related), malaria,pregnancy complications, and toxoplasmosis. DR4 polynucleotides orpolypeptides, or agonists or antagonists of DR4, can be used to treat,prevent and/or detect any of these symptoms or diseases.

In specific embodiments, DR4 polynucleotides, polypeptides, or agonistsand/or antagonists thereof are used to treat and/or prevent malaria.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are also useful as an adjuvant to enhanceimmune responsiveness to specific antigen, tumor-specific responses,and/or anti-viral immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immuneresponses that may be enhanced using the compositions of the inventionas an adjuvant, include virus and virus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g.,hepatitis B). In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response to avirus, disease, or symptom selected from the group consisting of:HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese Bencephalitis, Influenza A and B, Parainfluenza, Measles,Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpessimplex virus, and yellow fever.

Anti-bacterial or anti-fungal immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include bacteriaor fungus and bacteria or fungus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a bacterium or fungus, disease, or symptom selectedfrom the group consisting of: tetanus, diphtheria, botulism, andmeningitis type B. In another specific embodiment, the compositions ofthe invention are used as an adjuvant to enhance an immune response to abacteria selected from the group consisting of: Vibrio cholerae,Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Neisseriameningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigellaspp., Enterotoxigenic E. coli, Enterohemorrhagic E. coli, and Borreliaburgdorferi.

Anti-parasitic immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium spp. (malaria).

More generally, polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful in regulating(i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment and/or preventionof autoimmune disorders. In specific embodiments, polynucleotides and/orpolypeptides of the invention are used to treat and/or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or are otherwise known in the art.

In one embodiment, DR4 polynucleotides and/or polypeptides of theinvention and/or agonists or antagonists thereof may be used as animmune system enhancer prior to, during, or after bone marrow transplantand/or other transplants (e.g., allogenic or xenogenic organtransplantation). With respect to transplantation, compositions of theinvention may be administered prior to, concomitant with, and/or aftertransplantation. In a specific embodiment, compositions of the inventionare administered after transplantation, prior to the beginning ofrecovery of T-cell populations. In another specific embodiment,compositions of the invention are first administered aftertransplantation after the beginning of recovery of T-cell populations,but prior to full recovery of B-cell populations.

In another embodiment, DR4 polynucleotides and/or polypeptides of theinvention and/or agonists or antagonists thereof may be used as an agentto boost immunoresponsiveness among B-cell immunodeficient individuals.B-cell immunodeficiencies that may be ameliorated or treated and/orprevented by administering the DR4 polypeptides or polynucleotides ofthe invention, or agonists or antagonists thereof, include, but are notlimited to, severe combined immune deficiency (SCID), congenitalagammaglobulinemia, common variable immunodeficiency, Wiskott-AldrichSyndrome, and X-linked immunodeficiency with hyper IgM.

Additionally, DR4 polynucleotides and/or polypeptides of the inventionand/or agonists or antagonists thereof may be used as an agent to boostimmunoresponsiveness among individuals having an acquired loss of B-cellfunction. Conditions resulting in an acquired loss of B-cell functionthat may be ameliorated, treated, and/or prevented by administering theDR4 polypeptides or polynucleotides of the invention, or agonists orantagonists thereof, include, but are not limited to, HIV Infection,AIDS, bone marrow transplant, and B-cell chronic lymphocytic leukemia(CLL).

Furthermore, DR4 polynucleotides and/or polypeptides of the inventionand/or agonists or antagonists thereof may be used as an agent to boostimmunoresponsiveness among individuals having a temporary immunedeficiency. Conditions resulting in a temporary immune deficiency thatmay be ameliorated, treated, and/or prevented by administering the DR4polypeptides or polynucleotides of the invention, and/or agonists orantagonists thereof, include, but are not limited to, recovery fromviral infections (e.g., influenza), conditions associated withmalnutrition, recovery from infectious mononucleosis, or conditionsassociated with stress, recovery from measles, recovery from bloodtransfusion, recovery from surgery.

DR4 polynucleotides and/or polypeptides of the invention and/or agonistsor antagonists thereof may also be used as a regulator of antigenpresentation by monocytes, dendritic cells, and/or B-cells. In oneembodiment, DR4 (in soluble, membrane-bound or transmembrane forms)enhances antigen presentation or antagonizes antigen presentation invitro or in vivo.

In related embodiments, said enhancement or antagonization of antigenpresentation may be useful as an anti-tumor treatment or to modulate theimmune system. For example, DR4 polynucleotides and/or polypeptides ofthe invention and/or agonists or antagonists thereof may be used as anagent to direct an individual's immune system towards development of ahumoral response (i.e. TH2) as opposed to a TH1 cellular response. Also,DR4 polynucleotides and/or polypeptides of the invention and/or agonistsor antagonists thereof may be used as a stimulator of B-cell productionin pathologies such as AIDS, chronic lymphocyte disorder and/or CommonVariable Immunodeficiency.

In another embodiment, DR4 polynucleotides and/or polypeptides of theinvention and/or agonists or antagonists thereof may be used as a meansto induce tumor proliferation and thus make the tumor more susceptibleto anti-neoplastic agents. For example, multiple myeloma is a slowlydividing disease and is thus refractory to virtually all anti-neoplasticregimens. If these cells were forced to proliferate more rapidly theirsusceptibility profile would likely change.

Other embodiments where DR4 polynucleotides and/or polypeptides of theinvention and/or agonists or antagonists thereof may be used include,but are not limited to: as a stimulator of B-cell production inpathologies such as AIDS, chronic lymphocyte disorder and/or CommonVariable Immunodeficiency; as a therapy for generation and/orregeneration of lymphoid tissues following surgery, trauma or geneticdefect; as a gene-based therapy for genetically inherited disordersresulting in immuno-incompetence such as observed among SCID patients;as an antigen for the generation of antibodies to inhibit or enhance DR5mediated responses; as a means of activating T-cells; as pretreatment ofbone marrow samples prior to transplant (such treatment would increaseB-cell representation and thus accelerate recovery); as a means ofregulating secreted cytokines that are elicited by DR4; to modulate IgEconcentrations in vitro or in vivo; and to treat and/or preventIgE-mediated allergic reactions including, but are not limited to,asthma, rhinitis, and eczema.

Alternatively, DR4 polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment and/or preventionof autoimmune disorders. In specific embodiments, polynucleotides and/orpolypeptides of the invention are used to treat and/or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or are otherwise known in the art.

Preferably, treatment using DR4 polynucleotides or polypeptides, oragonists or antagonists of DR4, could either be by administering aneffective amount of DR4 polypeptide to the patient, or by removing cellsfrom the patient, supplying the cells with DR4 polynucleotide, andreturning the engineered cells to the patient (ex vivo therapy).Moreover, as further discussed herein, the DR4 polypeptide orpolynucleotide can be used as an adjuvant in a vaccine to raise animmune response against infectious disease.

Additional preferred embodiments of the invention include, but are notlimited to, the use of DR4 polypeptides and functional agonists orantagonists in the following applications: administration to an animal(e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig,chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate,and human, most preferably human) to boost the immune system to produceincreased quantities of one or more antibodies (e.g., IgG, IgA, IgM, andIgE), to induce higher affinity antibody production (e.g., IgG, IgA,IgM, and IgE), and/or to increase an immune response; or administrationto an animal (including, but not limited to, those listed above, andalso including transgenic animals) incapable of producing functionalendogenous antibody molecules or having an otherwise compromisedendogenous immune system, but which is capable of producing humanimmunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT Application Nos. WO98/24893, WO96/34096, WO96/33735, and WO91/10741.

Antagonists of DR4 include binding and/or inhibitory antibodies,antisense nucleic acids, ribozymes or soluble forms of the DR4receptor(s). These would be expected to reverse many of the activitiesof herein, as well as find clinical or practical application including,but not limited to the following applications. DR4 antagonists may beused as a means of blocking various aspects of immune responses toforeign agents or self, for example, autoimmune disorders such as lupus,and arthritis, as well as immunoresponsiveness to skin allergies,inflammation, bowel disease, injury and pathogens. Although our currentdata speaks directly to the potential role of DR4 in B-cell and T-cellrelated pathologies, it remains possible that other cell types may gainexpression or responsiveness to DR4. Thus, DR4 may, like CD40 and itsligand, may be regulated by the status of the immune system and themicroenvironment in which the cell is located. DR4 antagonists may beused as a therapy for preventing the B-cell proliferation and Igsecretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythematosus and; as aninhibitor of graft versus host disease or transplant rejection; as atherapy for B-cell malignancies such as ALL, Hodgkin's disease,non-Hodgkin's lymphoma, Chronic lymphocyte leukemia, plasmacytomas,multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases; as atherapy for chronic hypergammaglobulinemeia evident in such diseases asmonoclonalgammopathy of undetermined significance (MGUS), Waldenstrom'sdisease, related idiopathic monoclonalgammopathies, and plasmacytomas;as a therapy for decreasing cellular proliferation of Large B-cellLymphomas; as a means of decreasing the involvement of B-cells and Igassociated with Chronic Myelogenous Leukemia; or as an immunosuppressiveagent.

Furthermore, DR4 polypeptides or polynucleotides of the invention, orantagonists thereof may be used to modulate IgE concentrations in vitroor in vivo, or to treat and/or prevent IgE-mediated allergic reactionsincluding, but not limited to, asthma, rhinitis, and eczema.

All of the therapeutic applications of DR4 polynucleotides and/orpolypeptides of the invention and/or agonists and/or antagonists thereofdescribed herein may, in addition to their uses in human medicine, beused in veterinary medicine. The present invention includes treatment ofcompanion animals, including, but not limited to dogs, cats, ferrets,birds, and horses; food animals, including, but not limited to cows,pigs, chickens, and sheep; and exotic animals, e.g., zoo animals.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human, primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

DR4 polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof described herein may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., asdescribed herein.

In one aspect, the present invention is directed to a method forenhancing apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the DR4 polypeptide an effectiveamount of DR4 ligand, analog or an agonist capable of increasing DR4mediated signaling. Preferably, DR4 mediated signaling is increased totreat and/or prevent a disease wherein decreased apoptosis or decreasedcytokine and adhesion molecule expression is exhibited. An agonist caninclude soluble forms of DR4 and monoclonal antibodies directed againstthe DR4 polypeptide.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the, DR4 polypeptide aneffective amount of an antagonist capable of decreasing DR4 mediatedsignaling. Preferably, DR4 mediated signaling is decreased to treatand/or prevent a disease wherein increased apoptosis or NF-kB expressionis exhibited. An antagonist can include soluble forms of DR4 (e.g.,polypeptides containing all or a portion of the DR4 extracellulardomain) and monoclonal antibodies directed against the DR4 polypeptide.

By “agonist” is intended naturally occurring and synthetic compoundscapable of enhancing or potentiating apoptosis (e.g., stimulating DR4activities). By “antagonist” is intended naturally occurring andsynthetic compounds capable of inhibiting apoptosis (e.g., inhibitingDR4 activities). Whether any candidate “agonist” or “antagonist” of thepresent invention can enhance or inhibit apoptosis can be determinedusing art-known TNF-family ligand/receptor cellular response assays,including those described in more detail below.

The present invention further encompasses methods and compositions forkilling of cells expressing DR4 on their surface, comprising, oralternatively consisting of, contacting agonists of the invention withsuch cells expressing DR4 on their surface.

In preferred embodiments, the present invention further encompassesmethods and compositions for killing of cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, contactingagonistic anti-DR4 antibodies of the invention with such cellsexpressing DR4 on their surface.

In specific embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, contacting agonistsof the invention with such cells expressing DR4 on their surface.

In preferred embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, contactingagonistic anti-DR4 antibodies of the invention with such cellsexpressing DR4 on their surface.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising amino acids about 52 to about 184 of SEQ ID NO:2on their surface, comprising, or alternatively consisting of, contactingagonists of the invention with such cells expressing said polypeptide ontheir surface.

In preferred embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing a polypeptidecomprising amino acids about 52 to about 184 of SEQ ID NO:2 on theirsurface, comprising, or alternatively consisting of, contactingagonistic anti-DR4 antibodies of the invention with such cellsexpressing said polypeptide on their surface.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising the extracellular domain of the polypeptideencoded by the cDNA clone contained in ATCC Deposit No. 97920 on theirsurface, comprising, or alternatively consisting of, contacting agonistsof the invention with such cells expressing said polypeptide on theirsurface.

In preferred embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing a polypeptidecomprising the extracellular domain of the polypeptide encoded by thecDNA clone contained in ATCC Deposit No. 97920 on their surface,comprising, or alternatively consisting of, contacting agonisticanti-DR4 antibodies of the invention with such cells expressing saidpolypeptide on their surface.

The present invention further encompasses methods and compositions forkilling of cells expressing DR4 on their surface, comprising, oralternatively consisting of, administering to an animal, agonists of theinvention in an amount effective to kill such DR4 expressing cells.

In preferred embodiments, the present invention further encompassesmethods and compositions for killing of cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, administering to ananimal, agonistic anti-DR4 antibodies of the invention in an amounteffective to kill such DR4 expressing cells.

In specific embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, administering to ananimal, agonists of the invention in an amount effective to induceapoptosis in such DR4 expressing cells.

In preferred embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing DR4 on theirsurface, comprising, or alternatively consisting of, administering to ananimal, agonistic anti-DR4 antibodies of the invention in an amounteffective to induce apoptosis in such DR4 expressing cells.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising amino acids about 52 to about 184 of SEQ ID NO:2on their surface, comprising; or alternatively consisting of,administering to an animal, agonists of the invention in an amounteffective to induce apoptosis in such cells expressing said polypeptideon their surface.

In preferred embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing a polypeptidecomprising amino acids about 52 to about 184 of SEQ ID NO:2 on theirsurface, comprising, or alternatively consisting of, administering to ananimal, agonistic anti-DR4 antibodies of the invention in an amounteffective to induce apoptosis in such cells expressing said polypeptideon their surface.

In further specific embodiments, the present invention encompassesmethods and compositions for inducing apoptosis in cells expressing apolypeptide comprising the extracellular domain of the polypeptideencoded by the cDNA clone contained in ATCC Deposit No. 97920 on theirsurface, comprising, or alternatively consisting of, administering to ananimal, agonists of the invention in an amount effective to induceapoptosis in such cells expressing said polypeptide on their surface.

In preferred embodiments, the present invention encompasses methods andcompositions for inducing apoptosis in cells expressing a polypeptidecomprising the extracellular domain of the polypeptide encoded by thecDNA clone contained in ATCC Deposit No. 97920 on their surface,comprising, or alternatively consisting of, administering to an animal,agonistic anti-DR4 antibodies of the invention in an amount effective toinduce apoptosis in such cells expressing said polypeptide on theirsurface.

One such screening procedure involves the use of melanophores which aretransfected to express the receptor of the present invention. Such ascreening technique is described in PCT WO 92/01810, published Feb. 6,1992. Such an assay may be employed, for example, for screening for acompound which inhibits (or enhances) activation of the receptorpolypeptide of the present invention by contacting the melanophore cellswhich encode the receptor with both a TNF-family ligand and thecandidate antagonist (or agonist). Inhibition or enhancement of thesignal generated by the ligand indicates that the compound is anantagonist or agonist of the ligand/receptor signaling pathway.

Other screening techniques include the use of cells which express thereceptor (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation, for example, asdescribed in Science 246:181-296 (October 1989). For example, compoundsmay be contacted with a cell which expresses the receptor polypeptide ofthe present invention and a second messenger response, e.g., signaltransduction or pH changes, may be measured to determine whether thepotential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding thereceptor into Xenopus oocytes to transiently express the receptor. Thereceptor oocytes may then be contacted with the receptor ligand and acompound to be screened, followed by detection of inhibition oractivation of a calcium signal in the case of screening for compoundswhich are thought to inhibit activation of the receptor.

Another screening technique involves expressing in cells a constructwherein the receptor is linked to a phospholipase C or D. Such cellsinclude endothelial cells, smooth muscle cells, embryonic kidney cells,etc. The screening may be accomplished as herein above described bydetecting activation of the receptor or inhibition of activation of thereceptor from the phospholipase signal.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the receptor such that the cellexpresses the receptor on its surface and contacting the cell with acompound in the presence of a labeled form of a known ligand. The ligandcan be labeled, e.g., by radioactivity. The amount of labeled ligandbound to the receptors is measured, e.g., by measuring radioactivity ofthe receptors. If the compound binds to the receptor as determined by areduction of labeled ligand which binds to the receptors, the binding oflabeled ligand to the receptor is inhibited.

Further screening assays for agonist and antagonist of the presentinvention are described in Tartaglia, L. A., and Goeddel, D. V., J.Biol. Chem. 267(7):4304-4307 (1992).

Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular response to a TNF-family ligand. Themethod involves contacting cells which express the DR4 polypeptide witha candidate compound and a TNF-family ligand, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made with theligand in absence of the candidate compound, whereby an increasedcellular response over the standard indicates that the candidatecompound is an agonist of the ligand/receptor signaling pathway and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the ligand/receptor signalingpathway. By “assaying a cellular response” is intended qualitatively orquantitatively measuring a cellular response to a candidate compoundand/or a TNF-family ligand (e.g., determining or estimating an increaseor decrease in T-cell proliferation or tritiated thymidine labeling). Bythe invention, a cell expressing the DR4 polypeptide can be contactedwith either an endogenous or exogenously administered TNF-family ligand.

Agonist according to the present invention include naturally occurringand synthetic compounds such as, for example, TNF family ligand peptidefragments, transforming growth factor, neurotransmitters (such asglutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53),cytolytic T-cells and antimetabolites. Preferred agonist includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and β-amyloid peptide. (Science267:1457-1458 (1995)). Further preferred agonist include polyclonal andmonoclonal antibodies raised against the DR4 polypeptide, or a fragmentthereof. Such agonist antibodies raised against a TNF-family receptorare disclosed in Tartaglia, L. A., et al., Proc. Natl. Acad. Sci. USA88:9292-9296 (1991); and Tartaglia, L. A., and Goeddel, D. V., J. Biol.Chem. 267 (7):4304-4307 (1992). See, also, PCT Application WO 94/09137.

Antagonist according to the present invention include naturallyoccurring and synthetic compounds such as, for example, the CD40 ligand,neutral amino acids, zinc, estrogen, androgens, viral genes (such asAdenovirus ElB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barrvirus BHRF1, LMP-1, African swine fever virus LMW5-HL, and Herpesvirusyl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumorpromoters (such as PMA, Phenobarbital, and hexachlorocyclohexanes (e.g.,∀-, ∃-, or (-hexachlorocyclohexane).

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney etal., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991).The methods are based on binding of a polynucleotide to a complementaryDNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of the DR4receptor.

In one embodiment, the DR4 antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the DR4 antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding DR4, or fragments thereof, can be byany promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, Nature 29:304-310 (1981), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster et al., Nature 296:39-42(1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a DR4 gene.However, absolute complementarity, although preferred, is not required.A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case of doublestranded DR4 antisense nucleic acids, a single strand of the duplex DNAmay thus be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the larger thehybridizing nucleic acid, the more base mismatches with a DR4RNA it maycontain and still form a stable duplex (or triplex as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of the DR4 shown in SEQ IDNO:1 could be used in an antisense approach to inhibit translation ofendogenous DR4 mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of DR4 mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Nail. Acad. Sci. 84:648-652(1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual D-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the DR4 coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy DR4 mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of DR4 (SEQ ID NO:2). Preferably, the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the DR4 mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express DR4 invivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous DR4 messages and inhibit translation.Since ribozymes, unlike antisense molecules are catalytic, a lowerintracellular concentration is required for efficiency.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the DR4 gene and/or its promoter using targetedhomologous recombination. (e.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi(1987) and Thompson (1989), supra). However this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art. The contents of each of the documents recited in thisparagraph is herein incorporated by reference in its entirety.

Further antagonist according to the present invention include solubleforms of DR4, i.e., DR4 fragments that include the ligand binding domainfrom the extracellular region of the full length receptor. Such solubleforms of the receptor, which may be naturally occurring or synthetic,antagonize DR4 mediated signaling by competing with the cell surface DR4for binding to TNF-family ligands. Thus, soluble forms of the receptorthat include the ligand binding domain are novel cytokines capable ofinhibiting apoptosis induced by TNF-family ligands. These are preferablyexpressed as dimers or trimers, since these have been shown to besuperior to monomeric forms of soluble receptor as antagonists, e.g.,IgGFc-TNF receptor family fusions. Other such cytokines are known in theart and include Fas B (a soluble form of the mouse Fas receptor) thatacts physiologically to limit apoptosis induced by Fas ligand (Hughes,D. P. and Crispe, I. N., J. Exp. Med. 182:1395-1401 (1995)).

The experiments set forth in Example 5 demonstrates that DR4 is a deathdomain-containing molecule capable of triggering apoptosis which isimportant in the regulation of the immune system. In addition, theexperiments set forth below demonstrate that DR4-induced apoptosis wasblocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fink.Thus, inhibitors of ICE-like proteases, FADD-DN and FLICE-DN/MACHalC360S could also be used as antagonists for DR4 activity.

As discussed above, the term “antibody” (Ab) or “monoclonal antibody”(mAb) as used herein is meant to include intact molecules as well asfragments thereof (such as, for example, Fab and F(ab′)₂ fragments)which are capable of binding an antigen. Fab and F (ab′)₂ fragments lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J Nucl. Med. 24:316-325 (1983)).

Antibodies according to the present invention may be prepared by any ofa variety of methods using DR4 immunogens of the present invention. Asindicated, such DR4 immunogens include the full length (complete) DR4polypeptide (which may or may not include the leader sequence) and DR4polypeptide fragments such as the ligand binding domain, thetransmembrane domain, the intracellular domain and the death domain.

Proteins and other compounds which bind the DR4 domains are alsocandidate agonist and antagonist according to the present invention.Such binding compounds can be “captured” using the yeast two-hybridsystem (Fields and Song, Nature 340:245-246 (1989)). A modified versionof the yeast two-hybrid system has been described by Roger Brent and hiscolleagues (Gyuris, J. et al., Cell 75:791-803 (1993); Zervos, A. S. etal., Cell 72:223-232 (1993)). Preferably, the yeast two-hybrid system isused according to the present invention to capture compounds which bindto either the DR4 ligand binding domain or to the DR4 intracellulardomain. Such compounds are good candidate agonist and antagonist of thepresent invention.

By a “TNF-family ligand” is intended naturally occurring, recombinant,and synthetic ligands that are capable of binding to a member of the TNFreceptor family and inducing and/or blocking the ligand/receptorsignaling pathway. Members of the TNF ligand family include, but are notlimited to, DR4 ligands, TRAIL, TNF-α, TNF-β-∀, lymphotoxin-α (LT-α,also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β),FasL, VEGI (International Publication No. WO 96/14328), AIM-I(International Publication No. WO 97/33899), AIM-II (InternationalPublication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190),endokine-alpha (International Publication No. WO 98/07880),neutrokine-alpha (International Publication No. WO 98/18921), CD40L,CD27L, CD30L, 4-IBBL, OX40L and nerve growth factor (NGF).

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit and/or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the nucleic acid sequences include sequences encoding both the heavy andlight chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijistra et al., 1989, Nature 342:435-438).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors have been to delete retroviralsequences that are not necessary for packaging of the viral genome andintegration into host cell DNA. The nucleic acid sequences encoding theantibody to be used in gene therapy are cloned into one or more vectors,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300;U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asT-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see, e.g., PCT Publication WO 94/08598, dated Apr.28, 1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Modes of Administration

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

The agonist or antagonists described herein can be administered invitro, ex vivo, or in vivo to cells which express the receptor of thepresent invention. By administration of an “effective amount” of anagonist or antagonist is intended an amount of the compound that issufficient to enhance or inhibit a cellular response to a TNF-familyligand and include polypeptides. In particular, by administration of an“effective amount” of an agonist or antagonists is intended an amounteffective to enhance or inhibit DR4 mediated apoptosis. Of course, whereapoptosis is to be enhanced, an agonist according to the presentinvention can be co-administered with a TNF-family ligand. One ofordinary skill will appreciate that effective amounts of an agonist orantagonist can be determined empirically and may be employed in pureform or in pharmaceutically acceptable salt, ester or prodrug form. Theagonist or antagonist may be administered in compositions in combinationwith one or more pharmaceutically acceptable excipients.

It will be understood that, when administered to a human patient, thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon factors well known inthe medical arts.

As a general proposition, the total pharmaceutically effective amount ofDR4 polypeptide administered parenterally per dose will be in the rangeof about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the DR4 agonists or antagonists is typically administeredat a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by1-4 injections per day or by continuous subcutaneous infusions, forexample, using a mini-pump. An intravenous bag solution may also beemployed.

Dosaging may also be arranged in a patient specific manner to provide apredetermined concentration of an agonist or antagonist in the blood, asdetermined by the RIA technique. Thus patient dosaging may be adjustedto achieve regular on-going trough blood levels, as measured by RIA, onthe order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.

Pharmaceutical compositions of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use.

In addition to soluble DR4 polypeptides, DR4 polypeptide containing thetransmembrane region can also be used when appropriately solubilized byincluding detergents, such as CHAPS or NP-40, with buffer.

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In particular embodiments,pharmaceutical compositions are provided comprising an agonist orantagonist and a pharmaceutically acceptable carrier or excipient, whichmay be administered orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray.Importantly, by co-administering an agonist and a TNF-family ligand,clinical side effects can be reduced by using lower doses of both theligand and the agonist. It will be understood that the agonist can be“co-administered” either before, after, or simultaneously with theTNF-family ligand, depending on the exigencies of a particulartherapeutic application. By “pharmaceutically acceptable carrier” ismeant a non-toxic solid, semisolid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. In aspecific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion. The term“carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

As indicated above, the compositions of the invention may beadministered alone or in combination with other therapeutic agents.Therapeutic agents that may be administered in combination with thecompositions of the invention, include but are not limited to, othermembers of the TNF family, chemotherapeutic agents, antibiotics,steroidal and non-steroidal anti-inflammatories, conventionalimmunotherapeutic agents, cytokines, chemokines and/or growth factors.Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), (International Publication No. WO 96/14328), TNF-(-∀, TNF-(-∃(International Publication No. WO 00/08139), TRAIL, AIM-II(International Publication No. WO 97/34911), APRIL (J. Exp. Med.188(6):1185-1190), endokine-alpha (International Publication No. WO98/07880), TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), TR5 (InternationalPublication No. WO 98/30693), TR6 (International Publication No. WO98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9(International Publication No. WO 98/56892), TR10 (InternationalPublication No. WO 98/54202), 312C2 (International Publication No. WO98/06842), and TR12, AIM-I (International Publication No. WO 97/33899),and soluble forms CD154, CD70, and CD153.

In another embodiment, the compositions of the invention areadministered in combination with CD40 ligand (CD40L), a soluble form ofCD40L (e.g., AVRENDTA), biologically active fragments, variants, orderivatives of CD40L, anti-CD40L antibodies (e.g., agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonisticor antagonistic antibodies).

In yet another embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five, or more ofthe following compositions: tacrolimus (Fujisawa), thalidomide (e.g.,Celgene), anti-Tac(Fv)-PE40 (e.g., Protein Design Labs), inolimomab(Biotest), MAK-195F (Knoll), ASM-981 (Novartis), interleukin-1 receptor(e.g., Immunex), interleukin-4 receptor (e.g., Immunex), ICM3 (ICOS),BMS-188667 (Bristol-Myers Squibb), anti-TNF Ab (e.g., Therapeuticantibodies), CG-1088 (Celgene), anti-B7 monoclonal antibody (e.g.,Innogetics), MEDI-507 (BioTransplant), ABX-CBL (Abgenix).

According to the invention, a patient susceptible to both Fas ligand(Fas-L) mediated and TRAIL mediated cell death may be treated with bothan agent that inhibits TRAIL/TRAIL-R interactions and an agent thatinhibits Fas-L/Fas interactions. Suitable agents for blocking binding ofFas-L to Fas include, but are not limited to, soluble Fas polypeptides;oligomeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc);anti-Fas antibodies that bind Fas without transducing the biologicalsignal that results in apoptosis; anti-Fas-L antibodies that blockbinding of Fas-L to Fas; and muteins of Fas-L that bind Fas but do nottransduce the biological signal that results in apoptosis. Preferably,the antibodies employed according to this method are monoclonalantibodies. Examples of suitable agents for blocking Fas-L/Fasinteractions, including blocking anti-Fas monoclonal antibodies, aredescribed in WO 95/10540, hereby incorporated by reference.

In certain embodiments, compositions of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lanivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, compositions of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat and/or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, compositions of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat and/or prevent an opportunistic Mycobacteriumavium complex infection. In another specific embodiment, compositions ofthe invention are used in any combination with RIFABUTIN™,CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat and/orprevent an opportunistic Mycobacterium tuberculosis infection. Inanother specific embodiment, compositions of the invention are used inany combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ toprophylactically treat and/or prevent an opportunistic cytomegalovirusinfection. In another specific embodiment, compositions of the inventionare used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/orKETOCONAZOLE™ to prophylactically treat and/or prevent an opportunisticfungal infection. In another specific embodiment, compositions of theinvention are used in any combination with ACYCLOVIR™ and/orFAMCICOLVIR™ to prophylactically treat and/or prevent an opportunisticherpes simplex virus type I and/or type II infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylacticallytreat and/or prevent an opportunistic Toxoplasma gondii infection. Inanother specific embodiment, compositions of the invention are used inany combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylacticallytreat and/or prevent an opportunistic bacterial infection.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T-cells.

In specific embodiments, compositions of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the compositions of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In an additional embodiment, compositions of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the compositions of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,beta-lactamases, aminoglycosides, macrolides, quinolones,fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, andstreptomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In one embodiment, the compositions of the invention are administered incombination with steroid therapy. Steroids that may be administered incombination with the compositions of the invention, include, but are notlimited to, oral corticosteroids, prednisone, and methylprednisolone(e.g., IV methylprednisolone). In a specific embodiment, compositions ofthe invention are administered in combination with prednisone. In afurther specific embodiment, the compositions of the invention areadministered in combination with prednisone and an immunosuppressiveagent. Immunosuppressive agents that may be administered with thecompositions of the invention and prednisone are those described herein,and include, but are not limited to, azathioprine, cyclophosphamide, andcyclophosphamide IV. In a another specific embodiment, compositions ofthe invention are administered in combination with methylprednisolone.In a further specific embodiment, the compositions of the invention areadministered in combination with methylprednisolone and animmunosuppressive agent. Immunosuppressive agents that may beadministered with the compositions of the invention andmethylprednisolone are those described herein, and include, but are notlimited to, azathioprine, cyclophosphamide, and cyclophosphamide IV.

In another embodiment, the compositions of the invention areadministered in combination with an antimalarial. Antimalarials that maybe administered with the compositions of the invention include, but arenot limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In yet another embodiment, the compositions of the invention areadministered in combination with an NSAID.

In a nonexclusive embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five, ten, ormore of the following drugs: NRD-101 (Hoechst Marion Roussel),diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin(Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton(Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470(Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000(Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1 Ra genetherapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech),DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), soluble TNFreceptor 1 (synergen; Amgen), IPR-6001 (Institute for PharmaceuticalResearch), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals),BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim),LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), andbutixocort propionate (WarnerLambert).

In yet another embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five or more ofthe following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,auranofin, cyclosporine, penicillamine, azathioprine, an antimalarialdrug (e.g., as described herein), cyclophosphamide, chlorambucil, gold,ENBREL™ (Etanercept), anti-TNF antibody, and prednisolone. In a morepreferred embodiment, the compositions of the invention are administeredin combination with an antimalarial, methotrexate, anti-TNF antibody,ENBREL™ and/or suflasalazine.

In one embodiment, the compositions of the invention are administered incombination with methotrexate. In another embodiment, the compositionsof the invention are administered in combination with anti-TNF antibody.In another embodiment, the compositions of the invention areadministered in combination with methotrexate and anti-TNF antibody. Inanother embodiment, the compositions of the invention are administeredin combination with suflasalazine. In another specific embodiment, thecompositions of the invention are administered in combination withmethotrexate, anti-TNF antibody, and suflasalazine. In anotherembodiment, the compositions of the invention are administered incombination ENBREL™. In another embodiment, the compositions of theinvention are administered in combination with ENBREL™ and methotrexate.In another embodiment, the compositions of the invention areadministered in combination with ENBREL™, methotrexate andsuflasalazine. In another embodiment, the compositions of the inventionare administered in combination with ENBREL™, methotrexate andsuflasalazine. In other embodiments, one or more antimalarials iscombined with one of the above-recited combinations. In a specificembodiment, the compositions of the invention are administered incombination with an antimalarial (e.g., hydroxychloroquine), ENBREL™,methotrexate and suflasalazine. In another specific embodiment, thecompositions of the invention are administered in combination with anantimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNFantibody, and methotrexate.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); triterpenoids (e.g. oleanic acid and urosolic acid);cyclohexamide; casein kinase inhibitors; and others (e.g., dicarbazine,asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, andetoposide).

In specific embodiments, compositions of the present invention areadministered in combination with one or more chemotherapeutic agentsincluding, but not limited to, 81C6 (Anti-tenascin monoclonal antibody),2-chlorodeoxyadenosine, A007(4-4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone), Abarelix®(Abarelix-Depot-M®, PPI-149, R-3827); Abiraterone Acetate® (CB-7598,CB-7630), ABT-627 (ET-1 inhibitor), ABX-EGF (anti-EGFr MAb),Acetyldinaline (CI-994, GOE-5549, GOR-5549, PD-130636), AG-2034(AG-2024, AG-2032, GARFT [glycinamide ribonucleoside transformylase]inhibitor), Alanosine, Aldesleukin (IL-2, Proleukin®), Alemtuzumab®(Campath®), Alitretinoin (Panretin®, LGN-1057), Allopurinol (Aloprim®,Zyloprim®), Altretamine (Hexylen®), hexamethylmelamine, Hexastat®),Amifostine (Ethyol®), Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC603071), Aminoglutethimide (Cytadren®), Aminolevulinic acid (Levulan®,Kerastick®), Aminopterin, Amsacrine, Anastrozole (Arimidex®),Angiostatin, Annamycin (AR-522, annamycin LF, Aronex®), Anti-idiotypetherapy (BsAb), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv, anti-NHL MAb),APC-8015 (Provenge®, Dendritic cell therapy), Aplidine (Aplidin®,Aplidina®), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®, Compound506U78), Arsenic trioxide (Trisenox®, ATO, Atrivex®),Avorelin®(Meterelin®, MF-6001, EP-23904), B43-Genistein (anti-CD19Ab/genistein conjugate), B43-PAP (anti-CD19 Ab/pokeweed antiviralprotein conjugate), B7 antibody conjugates, BAY 43-9006 (Raf kinaseinhibitor), BBR 3464, Betathine (Beta-LT), Bevacizumab® (Anti-VEGFmonoclonal antibody, rhuMAb-VEGF), Bexarotene (Targretin®, LGD1069),BIBH-1 (Anti-FAP MAb), BIBX-1382, Biclutamide (Casodex®), Biricodardicitrate (Incel®, Incel MDR Inhibitor), Bleomycin (Blenoxane®), BLP-25(MUC-1 peptide), BLyS antagonists, BMS-214662 (BMS-192331, BMS-193269,BMS-206635), BNP-1350 (BNPI-1100, Karenitecins), BoronatedProtoporphyrin Compound (PDIT, Photodynamic Immunotherapy), Bryostatin-1(Bryostatin®, BMY-45618, NSC-339555), Budesonide (Rhinocort®), Busulfan(Busulfex®, Myleran®), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb,Cetuximab®), C242-DM1 (huC242-DM1), Cabergoline (Dostinex®),Capecitabine (Xeloda®, Doxifluridine®, oral 5-FU), Carbendazin®(FB-642), Carboplatin (Paraplatin®, CBDCA), Carboxyamidotriazole (NSC609974, CAI, L-651582), Carmustine (DTI-015, BCNU, BiCNU, GliadelWafer®), CC49-zeta gene therapy, CEA-cide® (Labetuzumab®, Anti-CEAmonoclonal antibody, hMN-14), CeaVac® (MAb 3H1), Celecoxib (Celebrex®),CEP-701 (KT-5555), Cereport® (Lobradimil®, RMP-7), Chlorambucil(Leukeran®), CHML (Cytotropic Heterogeneous Molecular Lipids),Cholecaliferol, CI-1033 (Pan-erbB RTK inhibitor), Cilengitide(EMD-121974, integrin alphavbeta3 antagonist), Cisplatin (Platinol®,CDDP), Cisplatin-epinephrine gel (IntraDose®, FocaCist®),Cisplatin-liposomal (SPI-077), 9-cis retinoic acid (9-cRA), Cladribine(2-CdA, Leustatin®), Clofarabine (chloro-fluoro-araA), Clonadinehydrochloride (Duraclon®), CMB-401 (Anti-PEM MAb/calicheamycin), CMT-3(COL-3, Metastat®), Cordycepin, Cotara® (chTNT-1/B, [¹³¹I]-chTNT-1/B),CN-706, CP-358774 (Tarceva®, OSI-774, EGFR inhibitor), CP-609754, CPIL-4-toxin (IL-4 fusion toxin), CS-682, CT-2584 (Apra®, CT-2583,CT-2586, CT-3536), CTP-37 (Avicine®, hCG blocking vaccine),Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine (Cytosar-U®,ara-C, cytosine arabinoside, DepoCyt®), D-limonene, DAB389-EGF (EGFfusion toxin), Dacarbazine (DTIC), Daclizumab® (Zenapax®), Dactinomycin(Cosmegen®), Daunomycin (Daunorubicin®, Cerubidine®), Daunorubicin(DaunoXome®, Daunorubicin®, Cerubidine®), DeaVac® (CEA anti-idiotypevaccine), Decitabine (5-aza-2′-deoxyytidine), Declopramide (Oxi-104),Denileukin diftitox (Ontak®), Depsipeptide (FR901228, FK228),Dexamethasone (Decadron®), Dexrazoxane (Zinecard®), Diethylnorspermine(DENSPM), Diethylstilbestrol (DES), Dihydro-5-azacytidine, Docetaxel(Taxotere®, Taxane®), Dolasetron mesylate (Anzemet®), Dolastatin-10(DOLA-10, NSC-376128), Doxorubicin (Adriamycin®, Doxil®, Rubex®), DPPE,DX-8951f (DX-8951), Edatrexate, EGF-P64k Vaccine, Elliott's B Solution®,EMD-121974, Endostatin, Eniluracil (776c85), EO9 (EO1, EO4, EO68, EO70,EO72), Epirubicin (Ellence®, EPI, 4′ epi-doxorubicin), Epratuzumab®(Lymphocide®, humanized anti-CD22, HAT), Erythropoietin (EPO®, Epogen®,Procrit®), Estramustine (Emcyt®), Etanidazole (Radinyl®), Etoposidephosphate (Etopophos®), Etoposide (VP-16, Vepesid®), Exemestane(Aromasin®, Nikidess®), Exetecan mesylate (DX-8951, DX-8951f), Exisulind(SAAND, Aptosyn®, cGMP-PDE2 and 5 inhibitor), F19 (Anti-FAP monoclonalantibody, iodinated anti-FAP MAb), Fadrozole (Afema®, Fadrozolehydrochloride, Arensin®), Fenretinide® (4HPR), Fentanyl citrate(Actiq®), Filgrastim (Neupogen®, G-CSF), FK-317 (FR-157471, FR-70496),Flavopiridol (HMR-1275), Fly3/flk2 ligand (Mobista®), Fluasterone,Fludarabine (Fludara®, FAMP), Fludeoxyglucose (F-18®), Fluorouracil(5-FU, Adrucil®, Fluoroplex®, Efudex®), Flutamide (Eulexin®), FMdC(KW-2331, MDL-101731), Formestane (Lentaron®), Fotemustine (Muphoran®,Mustophoran®), FUDR (Floxuridine®), Fulvestrant (Faslodex®), G3139(Genasense®, GentaAnticode®, Bcl-2 antisense), Gadolinium texaphyrin(Motexafin gadolinium, Gd-Tex®, Xcytrin®), Galarubicin hydrochloride(DA-125), GBC-590, Gastrimmune® (Anti-gastrin-17 immunogen, anti-g17),Gemcitabine (Gemto®, Gemzar®), Gentuzumab-ozogamicin (Mylotarg®), GL331,Globo H hexasaccharide (Globo H-KLH®), Glufosfamide®(β-D-glucosyl-isofosfamide mustard, D19575, INN), Goserelin acetate(Zoladex®), Granisetron (Xcytrin®), GVAX (GM-CSF gene therapy),Her-2/Neu vaccine, Herceptin® (Trastuzumab®, Anti-HER-2 monoclonalantibody, Anti-EGFR-2 MAb), HSPPC-96 (HSP cancer vaccine, gp96 heatshock protein-peptide complex), Hu1D10 (anti-HLA-DR MAb, SMART ID10),HumaLYM (anti-CD20 MAb), Hydrocortisone, Hydroxyurea (Hydrea®),Hypericin®(VIMRxyn®), 1-131 Lipidiol®, Ibritumomab® tiuxetan (Zevalin®),Idarubicin (Idamycin®, DMDR, IDA), Ifosfamide (IFEX®), Imatinib mesylate(STI-571, Imatinib®, Glivec®, Gleevec®, Ab1 tyrosine kinase inhibitor),INGN-101 (p53 gene therapy/retrovirus), INGN-201 (p53 genetherapy/adenovirus), Interferon alpha (Alfaferone®, Alpha-IF®),Interferon alpha 2a (Intron A®), Interferon gamma (Gamma-interferon,Gamma 100®, Gamma-IF), Interleukin-2 (ProleiukinR®), Intoplicine (RP60475), Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1),Irofulven (MGI-114, Ivofulvan, Acylfulvene analogue), ISIS-2053(PKC-alpha antisense), ISIS-2503 (Ras antisense), ISIS-3521 (PKC-alphaantisense), ISIS-5132 (K-ras/raf antisense), Isotretinoin (13-CRA,13-cis retinoic acid, Accutane®V), Ketoconazole (Nizoral®), KRN-8602(MX, MY-5, NSC-619003, MX-2), L-778123 (Ras inhibitors), L-asparaginase(Elspar®, Crastinin®, Asparaginase Medac®, Kidrolase®), Leflunomide(SU-101, SU-0200), Letrozole (Femara®), Leucovorin (Leucovorin®,Welicovorin®), Leuprolide acetate (Viadur®, Lupron®, Leuprogel®,Eligard®), Leuvectin® (cytofectin+IL-2 gene, IL-2 gene therapy),Levamisole (Ergamisol®), Liarozole (Liazal, Liazol, R-75251, R-85246,Ro-85264), Lmb-2 immunotoxin (anti-CD25 recombinant immuno toxin,anti-Tac(Fv)-PE38), Lometrexol (T-64, T-904064), Lomustine (CCNU®,CeeNU®), LY-335979, Lym-1 (131-1 LYM-1), Lymphoma vaccine (Genitope),Mannan-MUC1 vaccine, Marimastat® (BB-2516, TA-2516, MMP inhibitor),MDX-447 (MDX-220, BAB-447, EMD-82633, H-447, anti-EGFr/FcGammaR1r),Mechlorethamine (Nitrogen Mustard, HN₂, Mustargen®), Megestrol acetate(Megace®, Pallace®), Melphalan (L-PAM, Alkeran®, Phenylalanine mustard),Mercaptopurine (6-mercaptopurine, 6-MP), Mesna (Mesnex®), Methotrexate®(MTX, Mexate®, Folex®), Methoxsalen (Uvadex®), 2-Methoxyestradiol (2-ME,2-ME2), Methylprednisolone (Solumedrol®), Methyltestosterone(Android-10®, Testred®, Virilon®), MGV, Mitomycin C (Mitomycin®,Mutamycin®, Mito Extra®), Mitoxantrone (Novantrone®, DHAD), Mitumomab®(BEC-2, EMD-60205), Mivobulin isethionate (CI-980), MN-14 (Anti-CEAimmunoradiotherapy, ¹³¹ I-MN-14, ¹⁸⁸Re-MN-14), Motexafin Lutetium(Lutrin®, Optrin®, Lu-Tex®, lutetium texaphyrin, Lucyn®, Antrin®),MPV-2213ad (Finrozole®), MS-209, Muc-1 vaccine, NaPro Paclitaxel,Nelarabine (Compound 506, U78), Neovastat® (AE-941, MMP inhibitor),Neugene compounds (Oncomyc-NG, Resten-NG, myc antisense), Nilutamide(Nilandron®), NovoMAb-G2 scFv (NovoMAb-G2 IgM), NPI-0052 (proteasomeinhibitor), 06-benzylguanine (BG, Procept®), Octreotide acetate(Sandostatin LAR® Depot), Odansetron (Zofran®), Onconase (Ranpimase®),OncoVAX-CL, OncoVAX-CL Jenner (GA-733-2 vaccine), OncoVAX-P(OncoVAX-PrPSA), Onyx-015 (p53 gene therapy), Oprelvekin (Neumage®),Orzel (Tegafur+Uracil+Leucovorin), Oxaliplatin (Eloxatine®, Eloxatin®),Pacis® (BCG, live), Paclitaxel (Paxene®, Taxol®), Paclitaxel-DHA(Taxoprexin®), Pamidronate (Aredia®), PC SPES, Pegademase (Adagen®,Pegademase bovine), Pegaspargase®(Oncospar®), Peldesine (BCX-34, PNPinhibitor), Pemetrexed disodium (Alimta®, MTA, multitargeted antifolate,LY 231514), Pentostatin (Nipent®, 2-deoxycoformycin), Perfosfamide(4-hydroperoxycyclophosphamide, 4-HC), Perillyl alcohol (perillaalcohol, perillic alcohol, perillol, NSC-641066), Phenylbutyrate,Pirarubicin (THP), Pivaloyloxymethyl butyrate (AN-9, Pivanex®), Porfimersodium (Photofrin®), Prednisone, Prinomastat® (AG-3340, MMP inhibitor),Procarbazine (Matulane®), PROSTVAC, Providence Portland Medical CenterBreast Cancer Vaccine, PS-341 (LDP-341, 26S proteasome inhibitor), PSMAMAb (Prostate Specific Membrane Antigen monoclonal antibody),Pyrazoloacridine (NSC-366140, PD-115934), Quinine, R115777 (Zamestra®),Raloxifene hydrochloride (Evista®, Keoxifene hydrochloride), Raltitrexed(Tomudex®, ZD-1694), Rebeccamycin, Retinoic acid, R-flurbiprofen(Flurizan®, E-7869, MPC-7869), RFS-2000 (9-nitrocamptothecan, 9-NC,Rubitecan®), Rituximab®(Rituxan®, anti-CD20 MAb), RSR-13 (GSJ-61),Satraplatin (BMS-182751, JM-216), SCH 6636, SCH-66336, Sizofilan® (SPG,Sizofuran®, Schizophyllan®, Sonifilan®), SKI-2053R(NSC-D644591),Sobuzoxane (MST-16, Perazolin®), Squalamine (MSI-1256F), SR-49059(vasopressin receptor inhibitor, V1a), Streptozocin (Zanosar®), SU5416(Semaxanib®, VEGF inhibitor), SU6668 (PDGF-TK inhibitor), T-67(T-138067, T-607), Talc (Sclerosol®), Tamoxifen (Nolvadex®), Taurolidine(Taurolin®), Temozolamide (Temodar®, NSC 362856), Teniposide (VM-26,Vumon®)), TER-286, Testosterone (Andro®, Androderm®, Testoderm TTS®,Testoderm®, Depo-Testosterone®, Androgel®, depoAndro®), Tf-CRM107(Transferrin-CRM-107), Thalidomide and thalidomide analogs, includingbut not limited to, lenalidomide (CC-5013, REVLIMID®) and CC-4047(ACTIMID™), Theratope, Thioguanine (6-thioguanine, 6-TG), Thiotepa(triethylenethiophosphaoramide, Thioplex®), Thymosin alpha I (Zadaxin®,Thymalfasin®), Tiazofurin (Thiazole®), Tirapazamine (SR-259075, SR-4233,Tirazone®, Win-59075), TNP-470 (AGM-1470, Fumagillin), Tocladesine(8-Cl-cAMP), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®),Toremifene (Estrimex®, Fareston®), Tositumomab®g (Bexxar®), Tretinoin(Retin-A®, Atragen®, ATRA, Vesanoid®), TriAb® (anti-idiotype antibodyimmune stimulator), Trilostane (Modrefen®), Triptorelin pamoate(Trelstar Depot®, Decapeptyl®), Trimetrexate (Neutrexin®), Troxacitabine(BCH-204, BCH-4556, Troxatyl®), TS-1, UCN-01 (7-hydroxystaurosporine),Valrubicin (Valstar®), Valspodar (PSC 833), Vapreotide® (BMY-41606),Vaxid (B-cell lymphoma DNA vaccine), Vinblastine (Velban®V, VLB),Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®), Vindesine(Eldisine®g, Fildesin®), Vinorelbine (Navelbine®), Vitaxin® (LM-609,integrin alphavbeta3 antagonistic MAb), WF10 (macrophage regulator),WHI-P131, WT1 Vaccine, XR-5000 (DACA), XR-9576 (XR-9351,P-glycoprotein/MDR inhibitor), ZD-9331, ZD-1839 (IRESSA®), andZoledronate (Zometa®).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituximab and CHOP, or Rituximab and anycombination of the components of CHOP.

In further specific embodiments, compositions of the present inventionare administered in combination with one or more combinations ofchemotherapeutic agents including, but not limited to,9-aminocamptothecin+G-CSF, Adriamycin®+Blenoxane+Vinblastine+Dacarbazine(ABVD), BCNU (Carmustine)+Etoposide+Ara-C (Cytarabine)+Melphalen (BEAM),Bevacizumab®+Leucovorin, Bleomycin+Etoposide+Platinol® (Cisplatin)(BEP),Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+Vincristine+Procarbazine+Prednisone(BEACOPP), Bryostatin+Vincristine, Busulfan+Melphalan,Carboplatin+Cereport®, Carboplatin+Cyclophosphamide,Carboplatin+Paclitaxel, Carboplatin+Etoposide+Bleomycin (CEB),Carboplatin+Etoposide+Thiotepa, Cisplatin+Cyclophosphamide,Cisplatin+Docetaxel, Cisplatin+Doxorubicin, Cisplatin+Etoposide,Cisplatin+Gemcitabine, Cisplatin+Interferon alpha, Cisplatin+Irinotecan,Cisplatin+Paclitaxel, Cisplatin+Teniposide, Cisplatin+Vinblastine,Cisplatin+Vindesine, Cisplatin+Vinorelbine,Cisplatin+Cytarabine+Ifosfamide, Cisplatin+Ifosfamide+Vinblastine,Cisplatin+Vinblastine+Mitomycin C, Cisplatin+Vincristine+Fluorouracil,Cisplatin+Vincristine+Lomustine, Cisplatin+Vinorelbine+Gemcitabine,Cisplatin+Carmustine+Dacarbazine+Tamoxifen,Cisplatin+Cyclophosphamide+Etoposide+Vincristine, Cisplatin(Platinol®)+Oncovin®+Doxorubicin (Adriamycin®)+Etoposide (CODE),Cisplatin+Cytarabine+Ifosfamide+Etoposide+Methotrexate,Cyclophosphamide+Adriamycin® (Doxorubicin), Cyclophosphamide+Melphalan,Cyclophosphamide+SCH 6636, Cyclophosphamide+Adriamycin®+Cisplatin(Platinol®) (CAP), Cyclophosphamide+Adriamycing+Vincristine (CAV),Cyclophosphamide+Doxorubicin+Teniposide+Prednisone,Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon alpha,Cyclophosphamide+Epirubicin+Cisplatin (Platinol®) (CEP),Cyclophosphamide+Epirubicin+Fluorouracil,Cyclophosphamide+Methotrexate+Fluoruracil (CMF),Cyclophosphamide+Methotrexate+Vincristine (CMV),Cyclophosphamide+Adriamycin®+Methotrexate+Fluorouracil (CAMF),Cyclophosphamide+Adriamycing+Methotrexate+Procarbazine (CAMP),Cyclophosphamide+Adriamycin®+Vincristine+Etoposide (CAV-E),Cyclophosphamide+Adriamycin®+Vincristine+Prednisone (CHOP),Cyclophosphamide+Novantrone® (Mitoxantrone)+Vincristine(Oncovorin)+Prednisone (CNOP),Cyclophosphamide+Adriamycin®+Vincristine+Prednisone+Rituximab(CHOP+Rituximab), Cyclophosphamide+Adriamycing+Vincristine+Teniposide(CAV-T), Cyclophosphamide+Adriamycin®&+Vincristine alternating withPlatinol®+Etoposide (CAV/PE), Cyclophosphamide+BCNU (Carmustine)+VP-16(Etoposide) (CBV), Cyclophosphamide+Vincristine+Prednisone (CVP),Cyclophosphamide+Oncovin®+Methotrexate+Fluorouracil (COMF),Cytarabine+Methotrexate, Cytarabine+Bleomycin+Vincristine+Methotrexate(CytaBOM), Dactinomycin+Vincristine, Dexamethasone+Cytarabine+Cisplatin(DHAP), Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE),Docetaxel+Gemcitabine, Docetaxel+Vinorelbine,Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+Prednisone(Stanford V), Epirubicin+Gemcitabine, Estramustine+Docetaxel,Estramustine+Navelbine, Estramustine+Paclitaxel,Estramustine+Vinblastine, Etoposide (Vepesid®)+Ifosfamide+Cisplatin(Platinol®) (VIP), Etoposide+Vinblastine+Adriamycin (EVA), Etoposide(Vepesid®)+Ifosfamide+Cisplatin+Epirubicin (VIC-E),Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP),Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC),Fludarabine+Mitoxantrone+Dexamethasone (FMD),Fludarabine+Dexamethasone+Cytarabine (ara-C)+Cisplatin (Platinol™)(FluDAP), Fluorouracil+Bevacizumab®, Fluorouracil+CeaVac®,Fluorouracil+Leucovorin, Fluorouracil+Levamisole,Fluorouracil+Oxaliplatin, Fluorouracil+Raltitrexed, Fluorouracil+SCH6636, Fluorouracil+Trimetrexate, Fluorouracil+Leucovorin+Bevacizumab®,Fluorouracil+Leucovorin+Oxaliplatin,Fluorouracil+Leucovorin+Trimetrexate, Fluorouracil+Oncovin®+Mitomycin C(FOMi), Hydrazine+Adriamycin®+Methotrexate (HAM), Ifosfamide+Docetaxel,Ifosfamide+Etoposide, Ifosfamide+Gemcitabine, Ifosfamide+Paclitaxel,Ifosfamide+Vinorelbine, Ifosfamide+Carboplatin+Etoposide (ICE),Ifosfamide+Cisplatin+Doxorubicin, Irinotecan+C225 (Cetuximab®),Irinotecan+Docetaxel, Irinotecan+Etoposide, Irinotecan+Fluorouracil,Irinotecan+Gemcitabine, Mechlorethamine+Oncovin®(Vincristine)+Procarbazine (MOP), Mechlorethamine+Oncovin®(Vincristine)+Procarbazine+Prednisone (MOPP),Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate+Interferonalpha, Methotrexate+Vinblastine, Methotrexate+Cisplatin, Methotrexatewith leucovorinrescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone(m-BACOD), Mitomycin C+Ifosfamide+Cisplatin (Platinol®) (MIP), MitomycinC+Vinblastine+Paraplatin® (MVP), Mitoxantrone+Hydrocortisone,Mitoxantrone+Prednisone, Oncovin®+SCH 6636, Oxaliplatin+Leucovorin,Paclitaxel+Doxorubicin, Paclitaxel+SCH 6636, Paraplatin®g+Docetaxel,Paraplatin®+Etoposide, Paraplatin®+Gemcitabine, Paraplatin®+Interferonalpha, Paraplatin®+Irinotecan, Paraplatin®+Paclitaxel,Paraplatin®)+Vinblastine, Carboplatin (Paraplatin®)+Vincristine,Paraplatin®+Vindesine, Paraplatin®+Vinorelbine, Pemetrexeddisodium+Gemcitabine, Platinol® (Cisplatin)+Vinblastine+Bleomycin (PVB),Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide (ProMACE),Procarbazine+Lomustine, Procarbazine+Lomustine+Vincristine,Procarbazine+Lomustine+Vincristine+Thioguanine,Procarbazine+Oncovin®+CCNU®+Cyclophosphamide (POCC),Quinine+Doxorubicin, Quinine+Mitoxantrone+Cytarabine,Thiotepa+Etoposide, Thiotepa+Busulfan+Cyclophosphamide,Thiotepa+Busulfan+Melphalan, Thiotepa+Etoposide+Cannustine,Thiotepa+Etoposide+Carboplatin, Topotecan+Paclitaxel,Trimetrexate+Leucovorin, Vinblastine+Doxorubicin+Thiotepa,Vinblastine+Bleomycin+Etoposide+Carboplatin,Vincristine+Lomustine+Prednisone, Vincristine(Oncovin®)+Adriamycin®+Dexamethasone (VAD), Vincristine(Oncovin®)+Adriamycing+Procarbazine (VAP),Vincristine+Dactinomycin+Cyclophosphamide, and Vinorelbine+Gemcitabine.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13,IL-15, anti-CD40, CD40L, IFN-gamma and TNF-alpha.

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (P1GF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(P1GF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B 186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

In one embodiment, the compositions of the invention are administered incombination with one or more chemokines. In specific embodiments, thecompositions of the invention are administered in combination with anα(C×C) chemokine selected from the group consisting of gamma-interferoninducible protein-10 (γIP-10), interleukin-8 (IL-8), platelet factor-4(PF4), neutrophil activating protein (NAP-2), GRO-α, GRO-β, GRO-γ,neutrophil-activating peptide (ENA-78), granulocyte chemoattractantprotein-2 (GCP-2), and stromal cell-derived factor-1 (SDF-1, orpre-B-cell stimulatory factor (PBSF)); and/or a β (CC) selected from thegroup consisting of: RANTES (regulated on activation, normal T expressedand secreted), macrophage inflammatory protein-1 alpha (MIP-1α),macrophage inflammatory protein-1 beta (MIP-1β), monocyte chemotacticprotein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2), monocytechemotactic protein-3 (MCP-3), monocyte chemotactic protein-4 (MCP-4)macrophage inflammatory protein-1 gamma (MIP-1γ), macrophageinflammatory protein-3 alpha (MIP-3α), macrophage inflammatory protein-3beta (MIP-3 β), macrophage inflammatory protein-4 (MIP-4/DC-CK-1/PARC),eotaxin, Exodus, and 1-309; and/or the γ(C) chemokine, lymphotactin.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompounds or compositions may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol.Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al.,1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

In one embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing DR4 polypeptides or anti-DR4 antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells, expressing the membrane-boundform of DR4 on their surface. DR4 polypeptides or anti-DR4 antibodies ofthe invention may be associated with heterologous polypeptides,heterologous nucleic acids, toxins, or prodrugs via hydrophobic,hydrophilic, ionic and/or covalent interactions.

In one embodiment, the invention provides a method for the specificdelivery of compositions of the invention to cells by administeringpolypeptides of the invention (e.g., DR4 or anti-DR4 antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., DR4 polypeptides oranti-DR4 antibodies) in association with toxins or cytotoxic prodrugs.

In a specific embodiment, the invention provides a method for thespecific destruction of cells expressing DR4 receptors on their surface(e.g., activated T-cells, cancer cells, or leukemic cells) byadministering DR4 polypeptides in association with toxins or cytotoxicprodrugs.

In another specific embodiment, the invention provides a method for thespecific destruction of cells expressing the membrane-bound form of DR4on their surface (e.g., spleen, bone marrow, kidney and PBLs) byadministering anti-DR4 antibodies in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, cytotoxins (cytotoxic agents), or anymolecules or enzymes not normally present in or on the surface of a cellthat under defined conditions cause the cell's death. Toxins that may beused according to the methods of the invention include, but are notlimited to, radioisotopes known in the art, compounds such as, forexample, antibodies (or complement fixing containing portions thereof)that bind an inherent or induced endogenous cytotoxic effector system,thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin,Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin,pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” alsoincludes a cytostatic or cytocidal agent, a therapeutic agent or aradioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi,or other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label proteins (includingantibodies) of the invention. Such techniques include, but are notlimited to, the use of bifunctional conjugating agents (see, e.g., U.S.Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931;5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and5,808,003; the contents of each of which are hereby incorporated byreference in its entirety). A cytotoxin or cytotoxic agent includes anyagent that is detrimental to cells. Examples include paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

By “cytotoxic prodrug” is meant a non-toxic compound that is convertedby an enzyme, normally present in the cell, into a cytotoxic compound.Cytotoxic prodrugs that may be used according to the methods of theinvention include, but are not limited to, glutamyl derivatives ofbenzoic acid mustard alkylating agent, phosphate derivatives ofetoposide or mitomycin C, cytosine arabinoside, daunorubicin, andphenoxyacetamide derivatives of doxorubicin.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, M. et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide of theinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Chromosome Assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.The mapping of DNAs to chromosomes according to the present invention isan important first step in correlating those sequences with genesassociated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a DR4 gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA the is usedfor in situ chromosome mapping using well known techniques for thispurpose.

In addition, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes.

Fluorescence in situ hybridization (“FISH”) of a cDNA to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60.For a review of this technique, see Verma et al., Human Chromosomes: aManual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes)).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification in E. coli

The deposited cDNA encoding the mature DR4 protein (ATCC No. 97853) isamplified using PCR oligonucleotide primers specific to the aminoterminal sequences of the DR4 protein and to vector sequences 3′ to thegene. Additional nucleotides containing restriction sites to facilitatecloning are added to the 5′ and 3′ sequences respectively.

The following primers are used for expression of DR4 extracellulardomain in E. coli. 5′ primer 5′-GCGGCATGCATGATCAATCAATTGGCAC-3′ (SEQ IDNO:8) contains the underlined SphI site. 3′ primer:5′-GCGAAGCTTTCAATTATGTCCATTGCCTG-3′ (SEQ ID NO:9) contains theunderlined HindIII site. Vector is pQE60.

The restriction sites are convenient to restriction enzyme sites in thebacterial expression vector pQE60, which are used for bacterialexpression in these examples. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibioticresistance (“Ampr”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”).

The amplified DR4DNA and the vector pQE60 both are digested with SphIand HindIII and the digested DNAs are then ligated together. Insertionof the DDCR protein DNA into the restricted pQE60 vector places the DR4protein coding region downstream of and operably linked to the vector'sIPTG-inducible promoter and in-frame with an initiatingAUG/appropriately positioned for translation of DR4 protein.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”), isused in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressing DR4protein, is available commercially from Qiagen.

Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lad repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2× phosphate-buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2×PBS at a concentration of 95 μg/ml.

Example 2 Expression in Mammalian Cells

Most of the vectors used for the transient expression of a given genesequence in mammalian cells carry the SV40 origin of replication. Thisallows the replication of the vector to high copy numbers in cells(e.g., COS cells) which express the T antigen required for theinitiation of viral DNA synthesis. Any other mammalian cell line canalso be utilized for this purpose.

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, also cellular signals can be used (e.g., human actin,promoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12M1 (ATCC67109). Mammalian host cells that could be usedinclude, human HeLa, 283, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1 African green monkey cells, quail QC1-3cells, mouse L cells and Chinese hamster ovary cells.

Alternatively, a gene of interest can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) is a usefulmarker to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Using this marker, themammalian cells are grown in increasing amounts of methotrexate forselection and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) cells are often used for the production ofproteins.

The expression vectors pCI and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology438:44701 (1985)), plus a fragment of the CMV-enhancer (Boshart et al.,Cell 41:521-530 (1985)). Multiple cloning sites, e.g. with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of DR4 polypeptide. PlasmidpC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).The plasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., Alt, F. W., et al., J. Biol.Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C. Biochem. etBiophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.Biotechnology 9:64-68 (1991)). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Xba I,and ASP718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the DR4 polypeptide in a regulated way inmammalian cells (Goshen, M., & Bujard, H. Proc. Natl. Acad. Sci. USA89:5547-5551 (1992)). For the polyadenylation of the mRNA other signals,e.g., from the human growth hormone or globin genes can be used as well.Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete polypeptide is amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe desired portion of the gene. The 5′ primer containing the underlinedBamHI site, a Kozak sequence, and an AUG start codon, has the followingsequence: 5′ GCGGGATCCGCCATCATGGC GCCACCACCAGCTAGA 3′ (SEQ ID NO:10).The 3′ primer, containing the underlined BamHI site, has the followingsequence: 5′ GCGGGATCCTCACTCCAAGGACAC GGCAGAGCC 3′ (SEQ ID NO:11).

The amplified fragment is digested with the endonuclease BamHI and thenpurified again on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of Methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 3 Protein Fusions of DR4

DR4 polypeptides of the invention are optionally fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of DR4 polypeptides to His-tag,HA-tag, protein A, IgG domains, and maltose binding protein facilitatespurification. (See EP A 394,827; Traunecker, et al., Nature 331:84-86(1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases thehalf-life time in vivo. Nuclear localization signals fused to DR4polypeptides can target the protein to a specific subcellularlocalization, while covalent heterodimer or homodimers can increase ordecrease the activity of a fusion protein. Fusion proteins can alsocreate chimeric molecules having more than one function. Finally, fusionproteins can increase solubility and/or stability of the fused proteincompared to the non-fused protein. All of the types of fusion proteinsdescribed above can be made using techniques known in the art or byusing or routinely modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence described inSEQ ID NO:13. These primers also preferably contain convenientrestriction enzyme sites that will facilitate cloning into an expressionvector, preferably a mammalian expression vector.

For example, if the pC4 (Accession No. 209646) expression vector isused, the human Fc portion can be ligated into the BamHI cloning site.Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and DR4 polynucleotide, isolated by the PCR protocoldescribed in Example 1, is ligated into this BamHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Example 4 Cloning and Expression of the Soluble Extracellular Domain ofDR4 in a Baculovirus Expression System

The deposited cDNA encoding the soluble extracellular domain of DR4protein (ATCC No. 97853) is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

The 5′ primer for DR4 has the sequence: 5′ GCGGGATCCGCCATCATGGCGCCACCACCAGCTAGA 3′ (SEQ ID NO: 10) containing the underlined BamHIrestriction enzyme site. Inserted into an expression vector, asdescribed below, the 5′ end of the amplified fragment encoding DR4provides an efficient cleavage signal peptide. An efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in thevector portion of the construct.

The 3′ primer for both DR4 has the sequence: 5′ GCGGGATCCTCAATTATGTCCATTGCCTG 3′ (SEQ ID NO:12) containing the underlined BamHIrestriction followed by nucleotides complementary to the DR4 nucleotidesequence set out in SEQ ID NO:1, followed by the stop codon.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.)The fragment then is digested with BamHI and Asp718 and again ispurified on a 1% agarose gel.

The vector pA2 is used to express the DR4 protein in the baculovirusexpression system, using standard methods, such as those described inSummers et al., A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedron promoter of the Autograph californica nuclear polyhedrosisvirus (ACMNPV) followed by convenient restriction sites. For an easyselection of recombinant virus the beta-galactosidase gene from E. coliis inserted in the same orientation as the polyhedron promoter and isfollowed by the polyadenylation signal of the polyhedron gene. Thepolyhedron sequences are flanked at both sides by viral sequences forcell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like, such as anin-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170:31-39, among others.

The plasmid is digested with the restriction enzyme BamHI and then isdephosphorylated using calf intestinal phosphatase, using routineprocedures known in the art. The DNA is then isolated from a 1% agarosegel using a commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.).

Fragment and the dephosphorylated plasmid are ligated together with T4DNA ligase. E. coli HB101 cells are transformed with ligation mix andspread on culture plates. Bacteria are identified that contain theplasmid with the human DDCR gene by digesting DNA from individualcolonies using BamHI and then analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing. This plasmid is designated herein pBac DR4.

5 μg of the plasmid pBac DR4 is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBac DR4 are mixed in a sterile well of a microliter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27 E C. After 5 hours the transfection solution is removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. The plate is put back into an incubator and cultivationis continued at 27 E C for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 E C. A clonecontaining properly inserted DR4 is identified by DNA analysis includingrestriction mapping and sequencing. This is designated herein as V-DR4.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-DR4 at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 5 DR4 Induced Apoptosis in Mammalian Cells

Overexpression of Fas/APO-1 and TNFR-1 in mammalian cells mimicsreceptor activation (M. Muzio, et al., Cell 85, 817-827 (1996); M. P.Boldin, et al, Cell 85, 803-815 (1996)). Thus, this system was utilizedto study the functional role of DR4. Transient expression of DR4 in MCF7human breast carcinoma cells and 293 human embryonic kidney cellsinduced rapid apoptosis.

Cell death assays are performed essentially as previously described (A.M. Chinnaiyan, et al., Cell 81, 505-12 (1995); M. P. Boldin, et al, JBiol Chem 270, 7795-8 (1995); F. C. Kischkel, et al., EMBO 14, 5579-5588(1995); A. M. Chinnaiyan, et al., J Biol Chem 271, 4961-4965 (1996)).Briefly, MCF-7 human breast carcinoma clonal cell lines stablytransfected with either vector alone or a CrmA expression construct (M.Tewari, et al., J Biol Chem 270, 3255-60 (1995)), are transientlytransfected with pCMV-DR4-galactosidase (or pCMV-DR4-galactosidase(lacking the death domain)) in the presence of a ten-fold excess ofpcDNA3 expression constructs encoding the indicated proteins usinglipofectamine (GIBCO-BRL). 293 cells are likewise transfected using theCaPO₄ method. The ICE family inhibitor z-VAD-fmk (Enzyme SystemsProducts, Dublin, Calif.) is added to the cells at a concentration of 10μM, 5 hrs after transfection. 32 hours following transfection, cells arefixed and stained with X-Gal as previously described (A. M. Chinnaiyan,et al., Cell 81, 505-12 (1995); M. P. Boldin, et al., J Biol Chem 270,7795-8 (1995); F. C. Kischkel, et al., EMBO 14, 5579-5588 (1995)).

The cells displayed morphological alterations typical of cellsundergoing apoptosis, becoming rounded, condensed and detaching from thedish. Similar to TNFR-1 and Fas/APO-1 (M. Muzio, et al., Cell 85,817-827 (1996); M. P. Boldin, et al., Cell 85, 803-815 (1996); M.Tewari, et al., J Biol Chem 270, 3255-60 (1995)), DR4-induced apoptosiswas blocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk.

Example 6 The Extracellular Domain of DR4 Binds the Cytotoxic Ligand,TRAIL, and Blocks TRAIL-Induced Apoptosis

This example shows that the present receptor, DR4, binds TRAIL. Thesoluble extracellular ligand binding domain of DR4 was expressed as afusion to the Fc portion of human immunoglobulin (IgG). cDNA encodingthe extracellular domain of DR4 (amino acids 110 to 239) was obtained bypolymerase chain reaction and cloned into a modified pCMV1FLAG vectorthat allowed for in-frame fusion with the Fc portion of human IgG.

As shown in FIGS. 6A and 6B, DR4-Fc specifically bound TRAIL, but notthe related cytotoxic ligand TNFα. In this experiment, we transfected293 cells with constructs encoding the Fc-extracellular domains of DR4,TNFR1, or Fas, and the corresponding ligands, and the conditioned mediawas harvested 72 to 80 hours later, clarified by centrifugation,divided, and stored at −80° C. For binding assays, equal amounts ofreceptor-Fc- and ligand containing conditioned media were mixed inbuffer containing 50 mM Hepes, pH 7.0, 150 mM NaCl, 1 mM EDTA, 0.5%NP-40, and a protease inhibitor mixture, and the sample was incubated at4° C. with continuous rotation for 4 hours. Receptor-Fc-ligand complexeswere precipitated with protein G-Sepharose, extensively washed with theabove buffer, boiled in SDS sample buffer, and resolved on a 12%SDS-polyacrylamide gel and co-precipitated soluble ligands were detectedby immunoblotting with anti-Flag (Babco) or anti-myc-HRP (BMB) or toFasL (Pharmingen).

Additionally, DR4-Fc blocked the ability of TRAIL to induce apoptosis(FIG. 6A). MCF7 cells were treated with soluble TRAIL (400 ng/ml) in thepresence of equal amounts of Fc-fusions or Fc alone. Five hours latercells were fixed with formaldehyde, and the nuclei were stained with4′,6′-diamidino-2-phenylindole (DAPI) and examined by fluorescencemicroscopy with a fluorescein isothiocyanate range barrier filter cube.The data (mean 6±SD) shown in FIG. 6A are the percentage of apoptoticnuclei among total nuclei counted (n=3).

Finally, DR4-Fc had no effect on apoptosis TNFα-induced cell death underconditions where TNFR1-Fc completely abolished TNFα killing (FIG. 6B).MCF7 cells were treated with TNFα (40 ng/ml; Genentech, Inc.) in thepresence of equal amounts of Fc-fusions or Fc alone. Nuclei were stainedand examined 15 to 18 hours later.

Example 7 Assays to Detect Stimulation or Inhibition of B-cellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B-cell responsiveness including IL-2, IL-4, IL-5,IL6, IL-7, IL-10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B-cell populations.One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

Experimental Procedure

In Vitro assay-Purified DR4 protein, or truncated forms thereof, isassessed for its ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. The activity of DR4 protein on purified humantonsillar B-cells, measured qualitatively over the dose range from 0.1to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulationassay in which purified tonsillar B-cells are cultured in the presenceof either formalin-fixed Staphylococcus aureus Cowan I (SAC) orimmobilized anti-human IgM antibody as the priming agent. Second signalssuch as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicitB-cell proliferation as measured by tritiated-thymidine incorporation.Novel synergizing agents can be readily identified using this assay. Theassay involves isolating human tonsillar B-cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B-cells as assessed by expression of CD45R(B220).Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M βME, 100 U/ml penicillin, 10μg/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150μl. Proliferation or inhibition is quantitated by a 20 hour pulse (1μCi/well) with ³H-thymidine (6.7 Ci/mM) beginning 72 hour post factoraddition. The positive and negative controls are IL-2 and mediumrespectively.

In Vivo assay-BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of DR4 protein, or truncated forms thereof. Micereceive this treatment for 4 consecutive days, at which time they aresacrificed and various tissues and serum collected for analyses.Comparison of H&E sections from normal and DR4 protein-treated spleensidentify the results of the activity of DR4 protein on spleen cells,such as the diffusion of peri-arterial lymphatic sheaths, and/orsignificant increases in the nucleated cellularity of the red pulpregions, which may indicate the activation of the differentiation andproliferation of B-cell populations. Immunohistochemical studies using aB-cell marker, anti-CD45R (B220), are used to determine whether anyphysiological changes to splenic cells, such as splenic disorganization,are due to increased B-cell representation within loosely defined B-cellzones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from DR4 protein-treated mice isused to indicate whether DR4 protein specifically increases theproportion of ThB+, CD45R(B220)dull B-cells over that which is observedin control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andDR4 protein-treated mice.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 8 T-Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 μl/well of monoclonalantibody to CD3 (HIT3a, Pharmingen) or isotype-matched controlmonoclonal antibody (B33.1) overnight at 4° C. (1 μg/ml in 0.05Mbicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC areisolated by F/H gradient centrifugation from human peripheral blood andadded to quadruplicate wells (5×10⁴/well) of monoclonal antibody coatedplates in RPMI containing 10% FCS and P/S in the presence of varyingconcentrations of DR4 protein (total volume 200 iμl). Relevant proteinbuffer and medium alone are controls. After 48 hours culture at 37° C.,plates are spun for 2 minutes at 1000 rpm and 100 μl of supernatant isremoved and stored −20° C. for measurement of IL-2 (or other cytokines)if effect on proliferation is observed. Wells are supplemented with 100μl of medium containing 0.5 μCi of ³H-thymidine and cultured at 37° C.for 18-24 hr. Wells are harvested and incorporation of ³H-thymidine usedas a measure of proliferation. Anti-CD3 alone is the positive controlfor proliferation. IL-2 (100 U/ml) is also used as a control whichenhances proliferation. Control antibody which does not induceproliferation of T-cells is used as the negative controls for theeffects of DR4 proteins.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 9 Effect of DR4 on the Expression of MHC Class II, Costimulatoryand Adhesion Molecules and Cell Differentiation of monocytes andMonocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of DR4 or LPS (positivecontrol), washed with PBS containing 1% BSA and 0.02 mM sodium azide,and then incubated with 1:20 dilution of appropriate FITC- or PE-labeledmonoclonal antibodies for 30 minutes at 4° C. After an additional wash,the labeled cells are analyzed by flow cytometry on a FACScan (BectonDickinson).

Effect on the Production of Cytokines

Cytokines generated by dendritic cells, in particular IL-12, areimportant in the initiation of T-cell dependent immune responses. IL-12strongly influences the development of Th1 helper T-cell immuneresponse, and induces cytotoxic T and NK cell function. An ELISA is usedto measure the IL-12 release as follows. Dendritic cells (10⁶/ml) aretreated with increasing concentrations of DR4 for 24 hours. LPS (100ng/ml) is added to the cell culture as positive control. Supernatantsfrom the cell cultures are then collected and analyzed for IL-12 contentusing commercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)).The standard protocols provided with the kits are used.

Effect on the Expression of MHC Class II, Costimulatory and AdhesionMolecules

Three major families of cell surface antigens can be identified onmonocytes: adhesion molecules, molecules involved in antigenpresentation, and Fc receptor. Modulation of the expression of MHC classII antigens and other costimulatory molecules, such as B7 and ICAM-1,may result in changes in the antigen presenting capacity of monocytesand ability to induce T-cell activation. Increase expression of Fcreceptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of DR4 orLPS (positive control), washed with PBS containing 1% BSA and 0.02 mMsodium azide, and then incubated with 1:20 dilution of appropriate FITC-or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Monocyte Activation and/or Increased Survival:

Assays for molecules that activate (or alternatively, inactivate)monocytes and/or increase monocyte survival (or alternatively, decreasemonocyte survival) are known in the art and may routinely be applied todetermine whether a molecule of the invention functions as an inhibitoror activator of monocytes. DR4, agonists, or antagonists of DR4 can bescreened using the three assays described below. For each of theseassays, Peripheral blood mononuclear cells (PBMC) are purified fromsingle donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

1. Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from internally regulated process(apoptosis). Addition to the culture of activating factors, such asTNF-alpha dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of DR4. Cells are suspended at a concentrationof 2×10⁶/ml in PBS containing PI at a final concentration of 5 μg/ml,and then incubated at room temperature for 5 minutes before FAC Scananalysis. PI uptake has been demonstrated to correlate with DNAfragmentation in this experimental paradigm.

2. Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of DR4 and under the same conditions, butin the absence of DR4. For IL-12 production, the cells are primedovernight with IFN-γ (100 U/ml) in presence of DR4. LPS (10 ng/ml) isthen added. Conditioned media are collected after 24 h and kept frozenuntil use. Measurement of TNF-α, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit (e.g., R & D Systems(Minneapolis, Minn.)) applying the standard protocols provided with thekit.

3. Oxidative burst. Purified monocytes are plated in 96-well plate at2−1×10⁵ cell/well. Increasing concentrations of DR4 are added to thewells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS,glutamine and antibiotics). After 3 days incubation, the plates arecentrifuged and the medium is removed from the wells. To the macrophagemonolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mMpotassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol redand 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA).The plates are incubated at 37° C. for 2 hours and the reaction isstopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610nm. To calculate the amount of H₂O₂ produced by the macrophages, astandard curve of a H₂O₂ solution of known molarity is performed foreach experiment.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 10 The Effect of DR4 on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2−5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. DR4protein of SEQ ID NO. 2, and positive controls, such as VEGF and basicFGF (bFGF) are added, at varying concentrations. On days 4 and 6, themedium is replaced. On day 8, cell number is determined with a CoulterCounter.

An increase in the number of HUVEC cells after treatment with DR4indicates that DR4 may proliferate vascular endothelial cells.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 11 Stimulatory Effect of DR4 on the Proliferation of VascularEndothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)₂H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 ml serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or DR4 in 0.5% FBS) with or withoutHeparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMSmixture (1:0.05) are added per well and allowed to incubate for 1 hourat 37° C. before measuring the absorbance at 490 nm in an ELISA platereader. Background absorbance from control wells (some media, no cells)is subtracted, and seven wells are performed in parallel for eachcondition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518(1994).

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 12 Inhibition of PDGF-induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24hours, immunocytochemistry is performed by using BrdUrd Staining Kit(Zymed Laboratories). In brief, the cells are incubated with thebiotinylated mouse anti-BrdUrd antibody at 4° C. for 2 h after exposingto denaturing solution and then with the streptavidin-peroxidase anddiaminobenzidine. After counterstaining with hematoxylin, the cells aremounted for microscopic examination, and the BrdUrd-positive cells arecounted. The BrdUrd index is calculated as a percent of theBrdUrd-positive cells to the total cell number. In addition, thesimultaneous detection of the BrdUrd staining (nucleus) and the FITCuptake (cytoplasm) is performed for individual cells by the concomitantuse of bright field illumination and dark field-UV fluorescentillumination. See, Hayashida et al., J. Biol. Chem. 6;271(36):21985-21992 (1996).

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 13 Stimulation of Endothelial Migration

This example will be used to explore the possibility that DR4 maystimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., etal., “A 48 well micro chemotaxis assembly for rapid and accuratemeasurement of leukocyte migration.” J. Immunological Methods 33:239-247(1980)). Polyvinylpyrrolidone-free polycarbonate filters with a poresize of 8 μm (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1%gelatin for at least 6 hours at room temperature and dried under sterileair. Test substances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 μl of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 μl M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO₂ to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 14 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, DR4 activity can beassayed by determining nitric oxide production by endothelial cells inresponse to DR4. Nitric oxide is measured in 96-well plates of confluentmicrovascular endothelial cells after 24 hours starvation and asubsequent 4 hr exposure to various levels of a positive control (suchas VEGF-1) and DR4. Nitric oxide in the medium is determined by use ofthe Griess reagent to measure total nitrite after reduction of nitricoxide-derived nitrate by nitrate reductase. The effect of DR4 on nitricoxide release is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.). Calibration of the NO element isperformed according to the following equation:2KNO₂+2KI+2H₂SO₄62NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing K₁ and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas. The culture medium is removed and HUVECs arewashed twice with Dulbecco's phosphate buffered saline. The cells arethen bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) to maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 15 Effect of DR4 on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200μl/well) for 30 minutes at 37° C. CADMEC are seeded onto the coatedwells at 7,500 cells/well and cultured overnight in Growth Medium. TheGrowth Medium is then replaced with 300 μg Cell Applications ChordFormation Medium containing control buffer or DR4 (0.1 to 100 ng/ml) andthe cells are cultured for an additional 48 hr. The numbers and lengthsof the capillary-like chords are quantitated through use of theBoeckeler VIA-170 video image analyzer. All assays are done intriplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-estradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 16 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of DR4 to stimulate angiogenesis in CAMcan be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese quail (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old quailembryos is studied with the following methods.

On Day 4 of development, a window is made into the eggshell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors, and DR4, are dissolvedin distilled water and about 3.3 mg/5 ml are pipetted on the disks.After air-drying, the inverted disks are applied on CAM. After 3 days,specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi- and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 17 Angiogenesis Assay Using a Matrigel Implant in Mouse

In order to establish an in vivo model for angiogenesis to test DR4protein activities, mice and rats are implanted subcutaneously withmethylcellulose disks containing either 20 mg of BSA (negative control),1 mg of DR4, or 0.5 mg of VEGF-1 (positive control). The negativecontrol disks should contain little vascularization, while the positivecontrol disks should show signs of vessel formation.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 18 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of DR4 on ischemia, a rabbit hind-limbischemia model is created by surgical removal of one femoral arteries asdescribed previously (Takeshita, S. et al., Am J Pathol 147:1649-1660(1995)). The excision of the femoral artery results in retrogradepropagation of thrombus and occlusion of the external iliac artery.Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al., Am J Pathol 47:1649-1660 (1995)). An interval of10 days is allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb is transfected with 500 mg naked DR4expression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al., HumGene Ther. 4:749-758 (1993); Leclerc, G. et al., J. Clin. Invest. 90:936-944 (1992)). When DR4 is used in the treatment, a single bolus of500 mg DR4 protein or control is delivered into the internal iliacartery of the ischemic limb over a period of 1 minute through aninfusion catheter. On day 30, various parameters are measured in theserabbits: (a) BP ratio—The blood pressure ratio of systolic pressure ofthe ischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hind-limbs.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 19 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

A. Diabetic db+/db+ Mouse Model

To demonstrate that DR4 accelerates the healing process, the geneticallydiabetic mouse model of wound healing is used. The full thickness woundhealing model in the db+/db+ mouse is a well-characterized, clinicallyrelevant and reproducible model of impaired wound healing. Healing ofthe diabetic wound is dependent on formation of granulation tissue andre-epithelialization rather than contraction (Gartner, M. H. et al., J.Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol.136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67; (1980); Giacomelli et al., Lab Invest. 40(4):460-473(1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). Thesehomozygous diabetic mice develop hyperglycemia that is resistant toinsulin analogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andwere 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the covered by a continuous epithelium.

DR4 is administered using at a range different doses of DR4, from 4 mgto 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) DR4, 3) positive control.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day I was 64 mm², the corresponding size of the dermalpunch. Calculations were made using the following formula:[Open area on day8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of sections of bisected wounds.Histologic examination of the wounds are used to assess whether thehealing process and the morphologic appearance of the repaired skin isaltered by treatment with DR4. This assessment included verification ofthe presence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratincyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-mouse immune mouse IgG. Ranking of these sectionsis based on the extent of proliferation on a scale of 0-8, the lowerside of the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5:295-304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703-797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 1:703-797 (1978); Wahl, S. M., Glucocorticoids and woundhealing, In: Anti-inflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5:295-304(1991); Haynes, B. F., et al., J. Clin. Invest 1:703-797 (1978); Wahl,S. M., Glucocorticoids and wound healing, In: Anti-inflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989); Pierce, K. F. et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that DR4 can accelerate the healing process, the effectsof multiple topical applications of DR4 on full thickness excisionalskin wounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and were 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue was no longer visible andthe wound is covered by a continuous epithelium.

DR4 is administered using at a range different doses of DR4, from 4 mgto 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) were evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) DR4 treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1was 64 mm², the corresponding size of the dermal punch. Calculationswere made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining wasperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin was improved by treatmentwith DR4. A calibrated lens micrometer is used by a blinded observer todetermine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 20 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects ofDR4 in lymphangiogenesis and re-establishment of the lymphaticcirculatory system in the rat hind limb. Effectiveness is measured byswelling volume of the affected limb, quantification of the amount oflymphatic vasculature, total blood plasma protein, and histopathology.Acute lymphedema is observed for 7-10 days. Perhaps more importantly,the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70%ethanol. Blood is drawn for serum total protein testing. Circumferenceand volumetric measurements are made prior to injecting dye into pawsafter marking 2 measurement levels (0.5 cm above heel, at mid-pt ofdorsal paw). The intradermal dorsum of both right and left paws areinjected with 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then located and ligated by suturing.The popliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (A J Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software (Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs were amputated using a quillitine, thenboth experimental and control legs were cut at the ligature and weighed.A second weighing is done as the tibio-cacaneal joint was disarticulatedand the foot was weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80 EC until sectioning. Upon sectioning, the muscle wasobserved under fluorescent microscopy for lymphatics. Otherimmuno/histological methods are currently being evaluated.

The studies described in this example test the activity in DR4 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of DR4 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of DR4.

Example 21 Production of an Antibody

A. Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing DR4 are administered to an animal to inducethe production of sera containing polyclonal antibodies. In a preferredmethod, a preparation of DR4 protein is prepared and purified to renderit substantially free of natural contaminants. Such a preparation isthen introduced into an animal in order to produce polyclonal antiseraof greater specific activity.

Monoclonal antibodies specific for protein DR4 are prepared usinghybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler etal., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol.6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal(preferably a mouse) is immunized with DR4 polypeptide or, morepreferably, with a secreted DR4 polypeptide-expressing cell. Suchpolypeptide-expressing cells are cultured in any suitable tissue culturemedium, preferably in Earle's modified Eagle's medium supplemented with10% fetal bovine serum (inactivated at about 56° C.), and supplementedwith about 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the DR4 polypeptide.

Alternatively, additional antibodies capable of binding to DR4polypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the DR4protein-specific antibody can be blocked by DR4. Such antibodiescomprise anti-idiotypic antibodies to the DR4 protein-specific antibodyand are used to immunize an animal to induce formation of further DR4protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed infra. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

Isolation of Antibody Fragments Directed Against DR4-V1 and DR4 from aLibrary of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a large library of antibody fragments which contain reactivitiesagainst polypeptides of the present invention to which the donor may ormay not have been exposed (see, e.g., U.S. Pat. No. 5,885,793incorporated herein in its entirety by reference).

Rescue of the Library

A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO92/01047. To rescue phage displaying antibody fragments,approximately 10⁹ E. coli harboring the phagemid are used to inoculate50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin(2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of thisculture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×10⁸ TU of deltagene 3 helper phage (M13 gene 111, see WO92/01047) are added and theculture incubated at 37° C. for 45 minutes without shaking and then at37° C. for 45 minutes with shaking. The culture is centrifuged at 4000r.p.m. for 10 minutes and the pellet resuspended in 2 liters of 2×TYcontaining 100 μg/ml ampicillin and 50 μg/ml kanamycin and grownovernight. Phage are prepared as described in WO92/01047.

M13 gene III is prepared as follows: M13 gene III helper phage does notencode gene III protein, hence the phage(mid) displaying antibodyfragments have a greater avidity of binding to antigen. Infectious M13gene III particles are made by growing the helper phage in cellsharboring a pUC19 derivative supplying the wild type gene III proteinduring phage morphogenesis. The culture is incubated for 1 hour at 37°C. without shaking and then for a further hour at 37° C. with shaking.Cells are pelleted (IEC-Centra 8, 4000 revs/min for 10 min), resuspendedin 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μgkanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phageparticles are purified and concentrated from the culture medium by twoPEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS andpassed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100mg/ml or 10 mg/ml of a polypeptide of the present invention. Tubes areblocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 timesin PBS. Approximately 10¹³ TU of phage are applied to the tube andincubated for 30 minutes at room temperature tumbling on an over andunder turntable and then left to stand for another 1.5 hours. Tubes arewashed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage areeluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes onan under and over turntable after which the solution is immediatelyneutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used toinfect 10 ml of mid-log E. coli TG1 by incubating eluted phage withbacteria for 30 minutes at 37° C. The E. coli are then plated on TYEplates containing 1% glucose and 100 μg/ml ampicillin. The resultingbacterial library is then rescued with M13 gene III helper phage asdescribed above to prepare phage for a subsequent round of selection.This process is then repeated for a total of 4 rounds of affinitypurification with tube-washing increased to 20 times with PBS, 0.1%Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders

Eluted phage from the 3rd and 4th rounds of selection are used to infectE. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) fromsingle colonies for assay. ELISAs are performed with microtiter platescoated with either 10 pg/ml of the polypeptide of the present inventionin 50 mM bicarbonate pH 9.6. Clones positive in ELISA are furthercharacterized by PCR fingerprinting (see e.g., WO92/01047) and then bysequencing.

Example 22 Tissue distribution of DR4 Gene Expression

Northern blot analysis is carried out to examine DR4 gene (ATCC No.97853) expression in human tissues, using methods described by, amongothers, Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the DR4 protein (SEQ ID NO:1) is labeled with ³²Pusing the Rediprime™ DNA labeling system (Amersham Life Science),according to manufacturer's instructions. After labeling, the probe ispurified using a CHROMA SPIN-100™ column (Clontech Laboratories, Inc.),according to manufacturer's protocol number PT1200-1. The purifiedlabeled probe is then used to examine various human tissues for DR4mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with labeled probe using ExpressHyb™ hybridization solution(Clontech) according to manufacturer's protocol number PT1190-1.Following hybridization and washing, the blots are mounted and exposedto film at −70<C overnight, and films developed according to standardprocedures. Expression of DR4 was detected in tissues enriched inlymphocytes including amniotic cells, heart, liver cancer, kidney,peripheral blood leukocytes, activated T-cell, K562 plus PMA, W138cells, Th2 cells, human tonsils, and CD34 depleted buffy coat (cordblood). It can be envisaged that DR4 plays a role in lymphocytehomeostasis.

Example 23 Method of Determining Alterations in the DR4 Gene

RNA is isolated from entire families or individual patients presentingwith a phenotype of interest (such as a disease). cDNA is then generatedfrom these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 secondsat 52-58° C.; and 60-120 seconds at 70° C., using buffer solutionsdescribed in Sidransky, D., et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofDR4 are also determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations in DR4 is thencloned and sequenced to validate the results of the direct sequencing.

PCR products of DR4 are cloned into T-tailed vectors as described inHolton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156 (1991)and sequenced with T7 polymerase (United States Biochemical). Affectedindividuals are identified by mutations in DR4 not present in unaffectedindividuals.

Genomic rearrangements are also observed as a method of determiningalterations in the DR4 gene. Genomic clones isolated using techniquesknown in the art are nick-translated with digoxigenindeoxy-uridine5′-triphosphate (Boehringer Manheim), and FISH performed as described inJohnson, C. et al., Methods Cell Biol. 35:73-99 (1991). Hybridizationwith the labeled probe is carried out using a vast excess of human cot-1DNA for specific hybridization to the DR4 genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, C. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of DR4 (hybridized by the probe) areidentified as insertions, deletions, and translocations. These DR4alterations are used as a diagnostic marker for an associated disease.

Example 24 Method of Detecting Abnormal Levels of DR4 in a BiologicalSample

DR4 polypeptides can be detected in a biological sample, and if anincreased or decreased level of DR4 is detected, this polypeptide is amarker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect DR4 in asample, preferably a biological sample. Wells of a microtiter plate arecoated with specific antibodies to DR4, at a final concentration of 0.2to 10 μg/ml. The antibodies are either monoclonal or polyclonal and areproduced using technique known in the art. The wells are blocked so thatnon-specific binding of DR4 to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining DR4. Preferably, serial dilutions of the sample should beused to validate results. The plates are then washed three times withdeionized or distilled water to remove unbounded DR4.

Next, 50 μl of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate(NPP) substrate solution is then added to each well and incubated 1 hourat room temperature to allow cleavage of the substrate and fluorescence.The fluorescence is measured by a microtiter plate reader. A standardcurve is prepared using the experimental results from serial dilutionsof a control sample with the sample concentration plotted on the X-axis(log scale) and fluorescence or absorbance on the Y-axis (linear scale).The DR4 polypeptide concentration in a sample is then interpolated usingthe standard curve based on the measured fluorescence of that sample.

Example 25 Method of Treating Decreased Levels of DR4

The present invention relates to a method for treating an individual inneed of a decreased level of DR4 biological activity in the bodycomprising, administering to such an individual a composition comprisinga therapeutically effective amount of DR4 antagonist. Preferredantagonists for use in the present invention are DR4-specificantibodies.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of DR4 in an individual can betreated by administering DR4, preferably in a soluble and/or secretedform. Thus, the invention also provides a method of treatment of anindividual in need of an increased level of DR4 polypeptide comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of DR4 to increase the biological activity level ofDR4 in such an individual.

For example, a patient with decreased levels of DR4 polypeptide receivesa daily dose 0.1-100 μg/kg of the polypeptide for six consecutive days.Preferably, the polypeptide is in a soluble and/or secreted form.

Example 26 Method of Treating Increased Levels of DR4

The present invention also relates to a method for treating anindividual in need of an increased level of DR4 biological activity inthe body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of DR4 or an agonistthereof.

Antisense technology is used to inhibit production of DR4. Thistechnology is one example of a method of decreasing levels of DR4polypeptide, preferably a soluble and/or secreted form, due to a varietyof etiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels of DR4is administered intravenously antisense polynucleotides at 0.5, 1.0,1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated aftera 7-day rest period if the is determined to be well tolerated.

Example 27 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing soluble and/or mature DR4 polypeptides, onto a patient.Generally, fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37<C forapproximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding DR4 can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end encoding sequences respectively.Preferably, the 5′ primer contains an EcoRI site and the 3′ primerincludes a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the amplified EcoRI and HindIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is then used to transform E. coli HB101,which are then plated onto agar containing kanamycin for the purpose ofconfirming that the vector contains properly inserted DR4.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the DR4 gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the DR4 gene (the packaging cellsare now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a Millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether DR4 proteinis produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 28 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) DR4 sequences into an animal to increase ordecrease the expression of the DR4 polypeptide. The DR4 polynucleotidemay be operatively linked to a promoter or any other genetic elementsnecessary for the expression of the DR4 polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; Tabata H. et al., Cardiovasc. Res. 35:470-479 (1997); Chao J. et al., Pharmacol. Res. 35:517-522 (1997); WolffJ. A. Neuromuscul. Disord. 7:314-318 (1997); Schwartz B. et al., GeneTher. 3:405-411 (1996); Tsurumi Y. et al., Circulation 94:3281-3290(1996) (incorporated herein by reference).

The DR4 polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The DR4 polynucleotide constructscan be delivered in a pharmaceutically acceptable liquid or aqueouscarrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the DR4 polynucleotides may also be delivered inliposome formulations (such as those taught in Felgner P. L. et al. Ann.NY Acad. Sci. 772:126-139 (1995) and Abdallah B. et al. Biol. Cell85(1):1-7 (1995)) which can be prepared by methods well known to thoseskilled in the art.

The DR4 polynucleotide vector constructs used in the gene therapy methodare preferably constructs that will not integrate into the host genomenor will they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapies techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The DR4 polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked DR4 polynucleotide injection, an effective dosage amountof DNA or RNA will be in the range of from about 0.05 μg/kg body weightto about 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DR4polynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected DR4 polynucleotide in muscle invivo is determined as follows. Suitable DR4 template DNA for productionof mRNA coding for DR4 polypeptide is prepared in accordance with astandard recombinant DNA methodology. The template DNA, which may beeither circular or linear, is either used as naked DNA or complexed withliposomes. The quadriceps muscles of mice are then injected with variousamounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The DR4 template DNA is injected in 0.1 ml of carrier in a Icc syringe through a 27 gauge needle over one minute, approximately 0.5cm from the distal insertion site of the muscle into the knee and about0.2 cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 μmcross-section of the individual quadriceps muscles is histochemicallystained for DR4 protein expression. A time course for DR4 proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of DR4 DNAin muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using DR4 naked DNA.

Example 29 Sensitive and Specific Immunohistochemical Assays for theDetection of TRAIL Receptors DR4 and DR5

Specific and sensitive assays for the detection of DR4 and DR5 receptorsin formalin-fixed, paraffin-embedded human tumor tissues were developed.Affinity purified rabbit polyclonal antibodies, specific for DR4 andDR5, were optimized using a two step assay utilizing theEnVision®+visualization system. The optimal antigen retrieval wasdetermined to be heat induced epitope retrieval (HIER). The optimalantibody concentrations were established while employing an incubationtime of 30 minutes for the DR4 assay and 60 minutes for the DR5 assay.The reactions were visualized with 3′3-diaminobenzidine (DAB+).

Sections from formalin-fixed, paraffin-embedded pellets from cell linespreviously characterized by flow cytometry for surface expression of DR4and DR5 demonstrated optimal IHC staining using both assays. Similarly,tumor-enriched xenograft models showed IHC staining with minimalbackground staining of the surrounding non-reactive mouse tissue.Optimal staining for both DR4 and DR5 was observed on a wide range ofhuman tumor specimens, including colon, lung and breast carcinoma.Staining of the tumor cells was favorable with minimal backgroundstaining in the stroma.

Materials

Recombinant DR4 and DR5 Antigens:

DR4 and DR5 were expressed in NSO cells. The proteins were secreted inculture media. DR4 and DR5 were purified by a three-step procedurecombining HQ (PerSeptive Biosystems) anion exchange chromatography at pH8.5 using a salt gradient of 0-1 M NaCl) Hydrophobic InteractionChromatography on a Phenyl (high sub) Sepharose column (AmershamBioscience) and a Hydroxyapatite column for final purification andremoving high salt. Purified protein is endotoxin free and 99% pure inN-terminal sequencing and HPLC-RPC.

Primary antibody/recombinant antigen preparations in optimalconcentrations were mixed for 30 minutes at room temperature prior toapplication to the specimen.

Antibodies:

Affinity purified rabbit polyclonal antibodies specific for DR4 and DR5were produced by HGS. Optimal antibody titers were established usingcontrol specimens and diluted in Antibody Diluent with backgroundreducing components (DakoCytomation code S3022).

Target Retrieval:

Heat-induced epitope retrieval (HIER) was performed using targetretrieval solution, TRS (DakoCytomation code S1700) in a pressure cooker(Farberware Programmable Pressure Cooker #FPC400) for 30 minutes at highpressure.

Detection Reagents:

Immunostaining was performed using the EnVision+visualization system(DakoCytomation code K4011). This detection system utilizes an HRPenzyme labelled polymer backbone conjugated to mouse secondaryantibodies. The reactions were visualized with 3′3-diaminobenzidine(DAB+; DakoCytomation code K3468).

Cell Pellet Models:

Approximately one million cells were suspended in 100 L FACS buffer (PBSwith 0.1% sodium azide and 0.1% BSA) and incubated with PE-conjugatedantibodies directed against DR4 (eBiosciences #12-6644-73), DR5(eBiosciences #12-9908-73) or isotype matched control (eBiosciences#12-4719-71). Cells were incubated with antibodies for 10 to 20 minutesat room temperature, washed once in FACS buffer and pelleted bycentrifugation. The cells were resuspended in 0.5 μg/ml propidium iodidefor live/dead discrimination and analyzed on a FACScan using CellQuestsoftware (BD Immunocytometry Systems).

Control cell lines used were as follows: MDA-MB-231(breast carcinoma),Colo-205 (colon carcinoma), HT1080 (colon carcinoma), H460 (lungcarcinoma), H2122 (lung carcinoma) and ST486 (lymphoma). These controlcell lines were chosen based upon supportive flow cytometric, mRNAanalytical, in vitro cytotoxic response to agonist DR4 and DR5antibodies and/or protein chemical data to characterize expression ofboth DR4 and DR5. Four-micron sections of formalin-fixed,paraffin-embedded cell pellet blocks were mounted on silanized slides,deparaffinized with Histo-Clear (National Diagnostics) and rehydrated ingraded alcohol.

Xenograft Models:

Colo205 Colon: Female Swiss athymnic mice (7-8 weeks of age, 20 gaverage body weight) were used for xenograft models. On day 0, 1×107Colo205 cells (ATCC CCL-222) were implanted subcutaneously in the lowerright flank of the mice. Once tumors had grown to approximately 100 mm³(day 9) tumors were harvested.

MDA-MB-231 Breast: Female Swiss nude mice (7-10 weeks of age) were usedfor xenograft models. On day 0, 106 MDA-MB-231 cells (HGS stock) wereimplanted subcutaneously SC lower right mammary area (inguinal area) ofthe mice. Once tumors had grown to a approximately 25 mm³ (day 6) tumorswere harvested. Tumors were excised and placed in 10% neutral bufferedformalin (NBF) for 24 hours. Tumors were then trimmed, processed, andembedded in paraffin using routine histologic techniques. Four-micronsections of formalin-fixed, paraffin-embedded xenograft tissue blockswere mounted on silanized slides, deparaffinized with Histo-Clear(National Diagnostics) and rehydrated in graded alcohol.

Human Tissue Specimens: Four-micron sections of formalin-fixed,paraffin-embedded normal and neoplastic human tissue were mounted onsilanized slides. The tissue sections were deparaffinized in Histo-Clear(National Diagnostics) and rehydrated in graded alcohols. Some specimensused for the development of these assays used tissue samples provided bythe Cooperative Human Tissue Network which is funded by the NationalCancer Institute.

In conclusion, two highly specific IHC assays have been developed forthe detection of DR4 and DR5 expression in formalin-fixed,paraffin-embedded human tissues. Xenograft and cell pellet controls wereused to establish assay specificity. Optimal IHC staining for both DR4and DR5 was observed on a wide variety of formalin-fixed,paraffin-embedded human tumor tissues, including lung, colon and breastcarcinoma. DR5 tumor expression was stronger and more widespread thanDR4. Adjacent normal tissues have significantly weaker expression. Theobserved staining pattern was generally heterogeneous and eithermembrane or cytoplasmic in appearance. These assays allow for sensitiveand specific detection of DR4 and DR5 expression in human tissuesamples.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

Further, the Sequence Listing submitted herewith, in both computer andpaper forms, is hereby incorporated by reference in its entirety.

Additionally, the disclosures and sequence listings of U.S. ProvisionalApplication No. 60/990,687, filed Nov. 28, 2001, U.S. ProvisionalApplication No. 60/885,971, filed Jan. 22, 2007, U.S. patent applicationSer. No. 11/076,187, filed Mar. 10, 2005, U.S. Provisional ApplicationNo. 60/608,469, filed Sep. 10, 2004, U.S. Provisional Application No.60/551,768, filed Mar. 11, 2004, U.S. patent application Ser. No.10/648,786 filed Aug. 27, 2003, U.S. Provisional Application No.60/413,861 filed Sep. 27, 2002, U.S. Provisional Application No.60/406,922 filed Aug. 30, 2002, U.S. patent application Ser. No.09/565,918 filed May 5, 2000, now U.S. Pat. No. 6,433,137, U.S. patentapplication Ser. No. 09/448,868 filed Nov. 24, 1999, U.S. ProvisionalApplication No. 60/132,922 filed May 6, 1999, U.S. patent applicationSer. No. 09/013,895 filed Jan. 27, 1998, now U.S. Pat. No. 6,342,363,U.S. Provisional Application No. 60/037,829 filed Feb. 5, 1997, and U.S.Provisional Application No. 60/035,722 filed Jan. 28, 1997, are eachherein incorporated by reference in their entireties.

1. A method for inducing apoptosis of a DR4-expressing cell, comprisingcontacting said cell with an agonist antibody or fragment thereof thatspecifically binds to a polypeptide consisting of amino acids 24 to 238of SEQ ID NO:2 in combination with a second agent selected from thegroup consisting of: (a) an alkylating agent; (b) an antimetabolite; (c)a farnesyl transferase inhibitor; (d) a mitotic spindle inhibitor; (e) atopoisomerase inhibitor; (f) a tyrosine kinase inhibitor; (g) aproteasome inhibitor; (h) an antibiotic; (i) a histone deacetylaseinhibitor; (j) a HSP90 inhibitor; (k) thalidomide; (l) a thalidomideanalog; (m) a monoclonal antibody; (n) radiation therapy; (o) a AKT/mTORsignaling pathway inhibitor; and (p) a BCL-2 inhibitor.
 2. The method ofclaim 1, wherein said alkylating agent is a nitrogen mustard.
 3. Themethod of claim 1, wherein said alkylating agent is carboplatin.
 4. Themethod of claim 1, wherein said alkylating agent is cisplatin.
 5. Themethod of claim 1, wherein said alkylating agent is oxaliplatin.
 6. Themethod of claim 1, wherein said antimetabolite is methotrexate.
 7. Themethod of claim 1, wherein said antimetabolite isfluorouracil+leucovorin.
 8. The method of claim 1, wherein saidantimetabolite is cytosine arabinoside.
 9. The method of claim 1,wherein said antimetabolite is pemetrexed.
 10. The method of claim 1,wherein said antimetabolite is gemcitabine.
 11. The method of claim 1,wherein said antimetabolite is capecitabine.
 12. The method of claim 1,wherein said mitotic spindle inhibitor is docetaxel.
 13. The method ofclaim 1, wherein said mitotic spindle inhibitor is paclitaxel.
 14. Themethod of claim 1, wherein said mitotic spindle inhibitor isvincristine.
 15. The method of claim 1, wherein said mitotic spindleinhibitor is vinblastine.
 16. The method of claim 1, wherein saidtopoisomerase inhibitor is 9-aminocamptothecin.
 17. The method of claim1, wherein said topoisomerase inhibitor is topotecan.
 18. The method ofclaim 1, wherein said topoisomerase inhibitor is irinotecan.
 19. Themethod of claim 1, wherein said topoisomerase inhibitor is etoposide.20. The method of claim 1, wherein said tyrosine kinase inhibitor isimatinib mesylate.
 21. The method of claim 1, wherein said tyrosinekinase inhibitor is BAY 43-9006.
 22. The method of claim 1, wherein saidproteasome inhibitor is PS-341.
 23. The method of claim 1, wherein saidproteasome inhibitor is NPI-0052.
 24. The method of claim 1, whereinsaid antibiotic is bleomycin.
 25. The method of claim 1, wherein saidantibiotic is daunorubicin.
 26. The method of claim 1, wherein saidantibiotic is doxorubicin.
 27. The method of claim 1, wherein saidantibiotic is dactinomycin.
 28. The method of claim 1, wherein saidmonoclonal antibody is bevacizumab.
 29. The method of claim 1, whereinsaid monoclonal antibody is rituximab.
 30. The method of claim 1,wherein said thalidomide analog is revlimid.
 31. A method for treatingcancer, comprising administering to a patient therapeutically effectiveamounts of an agonist antibody or fragment thereof that specificallybinds to a polypeptide consisting of amino acids 24 to 238 of SEQ IDNO:2 in combination with a second agent selected from the groupconsisting of: (a) a tyrosine kinase inhibitor; (b) a proteasomeinhibitor; (c) an antibiotic; (d) a histone deacetylase inhibitor; (e) aHSP90 inhibitor; (f) thalidomide; (g) a thalidomide analog; (h) amonoclonal antibody; (i) radiation therapy; (j) a AKT/mTOR signalingpathway inhibitor; and (k) a BCL-2 inhibitor.
 32. The method of claim31, wherein said tyrosine kinase inhibitor is BAY 43-9006.
 33. Themethod of claim 31, wherein said proteasome inhibitor is NPI-0052. 34.The method of claim 31, wherein said antibiotic is bleomycin.
 35. Themethod of claim 31, wherein said antibiotic is daunorubicin.
 36. Themethod of claim 31, wherein said antibiotic is doxorubicin.
 37. Themethod of claim 31, wherein said antibiotic is dactinomycin.
 38. Themethod of claim 31, wherein said monoclonal antibody is bevacizumab. 39.The method of claim 31, wherein said monoclonal antibody is rituximab.40. The method of claim 31, wherein said thalidomide analog is revlimid.41. A method of treating cancer, comprising administering to a patient atherapeutically effective amount of an agonist antibody or fragmentthereof that specifically binds to a polypeptide consisting of aminoacids 24 to 238 of SEQ ID NO:2, wherein said cancer is selected from thegroup consisting of: (a) multiple myeloma; (b) bile duct cancer; (c)hepatoma; and (d) non-small cell lung cancer.
 42. The method of claim41, wherein said cancer is multiple myeloma.
 43. The method of claim 41,wherein said cancer is bile duct cancer.
 44. The method of claim 41,wherein said cancer is a hepatoma.
 45. The method of claim 41, whereinsaid cancer is non-small cell lung cancer.
 46. The method of claim 1,wherein said antibody or fragment thereof is a monoclonal antibody. 47.The method of claim 1, wherein said antibody or fragment thereof isselected from the group consisting of: (a) a polyclonal antibody; (b) achimeric antibody; (c) a human antibody; (d) a humanized antibody; (e) asingle-chain Fv antibody; and (f) an Fab antibody fragment.
 48. Themethod of claim 1, wherein said antibody or fragment thereof ispegylated.
 49. The method of claim 1, wherein said antibody or fragmentthereof is fused to a heterologous polypeptide.
 50. The method of claim1, wherein said cell is contacted by said antibody or fragment thereofand said second agent simultaneously.
 51. The method of claim 1, whereinsaid cell is contacted by said antibody or fragment thereof and saidsecond agent sequentially.
 52. The method of claim 1, which is in vitro.53. The method of claim 1, which is in vivo.
 54. The method of claim 1,wherein said cell is a cancer cell.
 55. The method of claim 31, whereinsaid antibody or fragment thereof is a monoclonal antibody.
 56. Themethod of claim 31, wherein said antibody or fragment thereof isselected from the group consisting of: (a) a polyclonal antibody; (b) achimeric antibody; (c) a human antibody; (d) a humanized antibody; (e) asingle-chain Fv antibody; and (f) an Fab antibody fragment.
 57. Themethod of claim 31, wherein said antibody or fragment thereof ispegylated.
 58. The method of claim 31, wherein said antibody or fragmentthereof is fused to a heterologous polypeptide.
 59. The method of claim31, wherein said antibody or fragment thereof and said second agent areadministered to the patient simultaneously.
 60. The method of claim 31,wherein said antibody or fragment thereof and said second agent areadministered to the patient sequentially.
 61. The method of claim 41,wherein said antibody or fragment thereof is a monoclonal antibody. 62.The method of claim 41, wherein said antibody or fragment thereof isselected from the group consisting of: (a) a polyclonal antibody; (b) achimeric antibody; (c) a human antibody; (d) a humanized antibody; (e) asingle-chain Fv antibody; and (f) an Fab antibody fragment.
 63. Themethod of claim 41, wherein said antibody or fragment thereof ispegylated.
 64. The method of claim 41, wherein said antibody or fragmentthereof is fused to a heterologous polypeptide.